Antisense modulation of apolipoprotein(a) expression

ABSTRACT

Antisense compounds, compositions and methods are provided for modulating the expression of apolipoprotein(a). The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding apolipoprotein(a). Methods of using these compounds for modulation of apolipoprotein(a) expression and for treatment of diseases associated with expression of apolipoprotein(a) are provided.

FIELD OF THE INVENTION

[0001] The present invention provides compositions and methods formodulating the expression of apolipoprotein(a). In particular, thisinvention relates to compounds, particularly oligonucleotides,specifically hybridizable with nucleic acids encoding apolipoprotein(a).Such compounds have been shown to modulate the expression ofapolipoprotein(a).

BACKGROUND OF THE INVENTION

[0002] Lipoproteins are globular, micelle-like particles that consist ofa non-polar core of acylglycerols and cholesteryl esters, surrounded byan amphiphilic coating consisting of protein, phospholipid andcholesterol. Lipoproteins have been classified into five broadcategories on the basis of their functional and physical properties:chylomicrons (which transport dietary lipids from intestine to tissues),very low density lipoproteins (VLDL), intermediate density lipoproteins(IDL), low density lipoproteins (LDL), (all of which transporttriacylglycerols and cholesterol from the liver to tissues), and highdensity lipoproteins (HDL) (which transport endogenous cholesterol fromtissues to the liver).

[0003] Lipoprotein particles undergo continuous metabolic processing andhave variable properties and compositions. Lipoprotein densitiesincrease without decreasing particle diameter because the density oftheir outer coatings is less than that of the inner core. The proteincomponents of lipoproteins are known as apolipoproteins. At least nineapolipoproteins are distributed in significant amounts among the varioushuman lipoproteins.

[0004] Lipoprotein(a) (also known as Lp(a)) is a cholesterol richparticle of the pro-atherogenic LDL class. Because Lp(a) is found onlyin Old World primates and European hedgehogs, it has been suggested thatit does not play an essential role in lipid and lipoprotein metabolism.Most studies have shown that high concentrations of Lp(a) are stronglyassociated with increased risk of cardiovascular disease (Rainwater andKammerer, J. Exp. Zool., 1998, 282, 54-61). These observations havestimulated numerous studies in humans and other primates to investigatethe factors that control Lp(a) concentrations and physiologicalproperties (Rainwater and Kammerer, J. Exp. Zool., 1998, 282, 54-61).

[0005] Lp(a) contains two disulfide-linked distinct proteins,apolipoprotein(a) (or ApoA) and apolipoprotein B (or ApoB) (Rainwaterand Kammerer, J. Exp. Zool., 1998, 282, 54-61). Apolipoprotein(a) is aunique apolipoprotein encoded by the LPA gene which has been shown toexclusively control the physiological concentrations of Lp(a) (Rainwaterand Kammerer, J. Exp. Zool., 1998, 282, 54-61). It varies in size due tointerallelic differences in the number of tandemly repeated Kringle4-encoding 5.5 kb sequences in the LPA gene (Rainwater and Kammerer, J.Exp. Zool., 1998, 282, 54-61).

[0006] Cloning of human apolipoprotein(a) in 1987 revealed homology tohuman plasminogen (McLean et al., Nature, 1987, 330, 132-137). The genelocus LPA encoding apolipoprotein(a) was localized to chromosome6q26-27, in close proximity to the homologous gene for plasminogen(Frank et al., Hum. Genet., 1988, 79, 352-356).

[0007] Transgenic mice expressing human apolipoprotein(a) were found tobe more susceptible than control mice to the development oflipid-staining lesions in the aorta and, consequently, apolipoprotein(a)is co-localized with lipid deposition in the artery walls (Lawn et al.,Nature, 1992, 360, 670-672). As an extension of these studies, it wasestablished that the major in vivo action of apolipoprotein(a) isinhibition of the conversion of plasminogen to plasmin which causesdecreased activation of latent transforming growth factor-beta. Becausetransforming growth factor-beta is a negative regulator of smooth musclecell migration and proliferation, inhibition of plasminogen activationindicates a possible mechanism for apolipoprotein(a) induction ofatherosclerotic lesions (Grainger et al., Nature, 1994, 370, 460-462).

[0008] Elevated plasma levels of Lp(a), caused by increased expressionof apolipoprotein(a), are associated with increased risk foratherosclerosis and its manifestations, which includehypercholesterolemia (Seed et al., N. Engl. J. Med. 1990, 322,1494-1499), myocardial infarction (Sandkamp et al., Clin. Chem., 1990,36, 20-23), and thrombosis (Nowak-Gottl et al., Pediatrics, 1997, 99,Ell).

[0009] Moreover, the plasma concentration of Lp(a) is stronglyinfluenced by heritable factors and is refractory to most drug anddietary manipulation (Katan and Beynen, Am. J. Epidemiol., 1987, 125,387-399; Vessby et al., Atherosclerosis, 1982, 44, 61-71). Pharmacologictherapy of elevated Lp(a) levels has been only modestly successful andapheresis remains the most effective therapeutic modality (Hajjar andNachman, Annu. Rev. Med., 1996, 47, 423-442).

[0010] Morishita et al. have reported the use of ribozymeoligonucleotides against apolipoprotein(a) for inhibition ofapolipoprotein(a) expression in HepG2 cells (Morishita et al.,Circulation, 1998, 98, 1898-1904).

[0011] Disclosed and claimed in U.S. Pat. No. 5,721,138 are nucleotidesequences encoding the human apolipoprotein(a) gene 5′-regulatory regionand isolated nucleotide sequences comprising at least thirty consecutivecomplementary nucleotides from human apolipoprotein(a) from nucleotideposition -208 to -1448 (Lawn, 1998).

[0012] To date, investigative and therapeutic strategies aimed atinhibiting apolipoprotein(a) function have involved the previously citeduse of Lp(a) apheresis and ribozyme oligonucleotides. Consequently,there remains a long-felt need for additional agents capable ofeffectively inhibiting apolipoprotein(a) function.

[0013] Antisense technology is emerging as an effective means ofreducing the expression of specific gene products and may thereforeprove to be uniquely useful in a number of therapeutic, diagnostic andresearch applications involving modulation ofapolipoprotein(a)expression.

[0014] The present invention provides compositions and methods formodulating apolipoprotein(a) expression.

SUMMARY OF THE INVENTION

[0015] The present invention is directed to compounds, particularlyantisense oligonucleotides, which are targeted to a nucleic acidencoding apolipoprotein(a), and which modulate the expression ofapolipoprotein(a). Pharmaceutical and other compositions comprising thecompounds of the invention are also provided. Further provided aremethods of modulating the expression of apolipoprotein(a) in cells ortissues comprising contacting said cells or tissues with one or more ofthe antisense compounds or compositions of the invention. Furtherprovided are methods of treating an animal, particularly a human,suspected of having or being prone to a disease or condition associatedwith expression of apolipoprotein(a), by administering a therapeuticallyor prophylactically effective amount of one or more of the antisensecompounds or compositions of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention employs oligomeric compounds, particularlyantisense oligonucleotides, for use in modulating the function ofnucleic acid molecules encoding apolipoprotein(a), ultimately modulatingthe amount of apolipoprotein(a) produced. This is accomplished byproviding antisense compounds which specifically hybridize with one ormore nucleic acids encoding apolipoprotein(a). As used herein, the terms“target nucleic acid” and “nucleic acid encoding apolipoprotein(a)”encompass DNA encoding apolipoprotein(a), RNA (including pre-mRNA andmRNA) transcribed from such DNA, and also cDNA derived from such RNA.The specific hybridization of an oligomeric compound with its targetnucleic acid interferes with the normal function of the nucleic acid.This modulation of function of a target nucleic acid by compounds whichspecifically hybridize to it is generally referred to as “antisense”.The functions of DNA to be interfered with include replication andtranscription. The functions of RNA to be interfered with include allvital functions such as, for example, translocation of the RNA to thesite of protein translation, translation of protein from the RNA,splicing of the RNA to yield one or more mRNA species, and catalyticactivity which may be engaged in or facilitated by the RNA. The overalleffect of such interference with target nucleic acid function ismodulation of the expression of apolipoprotein(a). In the context of thepresent invention, “modulation” means either an increase (stimulation)or a decrease (inhibition) in the expression of a gene. In the contextof the present invention, inhibition is the preferred form of modulationof gene expression and mRNA is a preferred target.

[0017] It is preferred to target specific nucleic acids for antisense.“Targeting” an antisense compound to a particular nucleic acid, in thecontext of this invention, is a multi step process. The process usuallybegins with the identification of a nucleic acid sequence whose functionis to be modulated. This may be, for example, a cellular gene (or mRNAtranscribed from the gene) whose expression is associated with aparticular disorder or disease state, or a nucleic acid molecule from aninfectious agent. In the present invention, the target is a nucleic acidmolecule encoding apolipoprotein(a). The targeting process also includesdetermination of a site or sites within this gene for the antisenseinteraction to occur such that the desired effect, e.g., detection ormodulation of expression of the protein, will result. Within the contextof the present invention, a preferred intragenic site is the regionencompassing the translation initiation or termination codon of the openreading frame (ORF) of the gene. Since, as is known in the art, thetranslation initiation codon is typically 5′-AUG (in transcribed mRNAmolecules; 5′-ATG in the corresponding DNA molecule), the translationinitiation codon is also referred to as the “AUG codon,” the “startcodon” or the “AUG start codon”. A minority of genes have a translationinitiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, theterms “translation initiation codon” and “start codon” can encompassmany codon sequences, even though the initiator amino acid in eachinstance is typically methionine (in eukaryotes) or formylmethionine (inprokaryotes). It is also known in the art that eukaryotic andprokaryotic genes may have two or more alternative start codons, any oneof which may be preferentially utilized for translation initiation in aparticular cell type or tissue, or under a particular set of conditions.In the context of the invention, “start codon” and “translationinitiation codon” refer to the codon or codons that are used in vivo toinitiate translation of an mRNA molecule transcribed from a geneencoding apolipoprotein(a), regardless of the sequence(s) of suchcodons.

[0018] It is also known in the art that a translation termination codon(or “stop codon”) of a gene may have one of three sequences, i.e.,5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA,5′-TAG and 5′-TGA, respectively). The terms “start codon region” and“translation initiation codon region” refer to a portion of such an mRNAor gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationinitiation codon. Similarly, the terms “stop codon region” and“translation termination codon region” refer to a portion of such anmRNA or gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationtermination codon.

[0019] The open reading frame (ORF) or “coding region,” which is knownin the art to refer to the region between the translation initiationcodon and the translation termination codon, is also a region which maybe targeted effectively. Other target regions include the 5′untranslated region (5′UTR), known in the art to refer to the portion ofan mRNA in the 5′ direction from the translation initiation codon, andthus including nucleotides between the 5′ cap site and the translationinitiation codon of an mRNA or corresponding nucleotides on the gene,and the 3′ untranslated region (3′UTR), known in the art to refer to theportion of an mRNA in the 3′ direction from the translation terminationcodon, and thus including nucleotides between the translationtermination codon and 3′ end of an mRNA or corresponding nucleotides onthe gene. The 5′ cap of an mRNA comprises an N7-methylated guanosineresidue joined to the 5′-most residue of the mRNA via a 5′-5′triphosphate linkage. The 5′ cap region of an mRNA is considered toinclude the 5′ cap structure itself as well as the first 50 nucleotidesadjacent to the cap. The 5′ cap region may also be a preferred targetregion.

[0020] Although some eukaryotic mRNA transcripts are directlytranslated, many contain one or more regions, known as “introns,” whichare excised from a transcript before it is translated. The remaining(and therefore translated) regions are known as “exons” and are splicedtogether to form a continuous mRNA sequence. mRNA splice sites, i.e.,intron-exon junctions, may also be preferred target regions, and areparticularly useful in situations where aberrant splicing is implicatedin disease, or where an overproduction of a particular mRNA spliceproduct is implicated in disease. Aberrant fusion junctions due torearrangements or deletions are also preferred targets. It has also beenfound that introns can also be effective, and therefore preferred,target regions for antisense compounds targeted, for example, to DNA orpre-mRNA.

[0021] Once one or more target sites have been identified,oligonucleotides are chosen which are sufficiently complementary to thetarget, i.e., hybridize sufficiently well and with sufficientspecificity, to give the desired effect.

[0022] In the context of this invention, “hybridization” means hydrogenbonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary nucleoside or nucleotide bases.For example, adenine and thymine are complementary nucleobases whichpair through the formation of hydrogen bonds. “Complementary,” as usedherein, refers to the capacity for precise pairing between twonucleotides. For example, if a nucleotide at a certain position of anoligonucleotide is capable of hydrogen bonding with a nucleotide at thesame position of a DNA or RNA molecule, then the oligonucleotide and theDNA or RNA are considered to be complementary to each other at thatposition. The oligonucleotide and the DNA or RNA are complementary toeach other when a sufficient number of corresponding positions in eachmolecule are occupied by nucleotides which can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termswhich are used to indicate a sufficient degree of complementarity orprecise pairing such that stable and specific binding occurs between theoligonucleotide and the DNA or RNA target. It is understood in the artthat the sequence of an antisense compound need not be 100%complementary to that of its target nucleic acid to be specificallyhybridizable. An antisense compound is specifically hybridizable whenbinding of the compound to the target DNA or RNA molecule interfereswith the normal function of the target DNA or RNA to cause a loss ofutility, and there is a sufficient degree of complementarity to avoidnon-specific binding of the antisense compound to non-target sequencesunder conditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and in the case of in vitro assays, under conditions in whichthe assays are performed.

[0023] Antisense and other compounds of the invention which hybridize tothe target and inhibit expression of the target are identified throughexperimentation, and the sequences of these compounds are hereinbelowidentified as preferred embodiments of the invention. The target sitesto which these preferred sequences are complementary are hereinbelowreferred to as “active sites” and are therefore preferred sites fortargeting. Therefore another embodiment of the invention encompassescompounds which hybridize to these active sites.

[0024] Antisense compounds are commonly used as research reagents anddiagnostics. For example, antisense oligonucleotides, which are able toinhibit gene expression with exquisite specificity, are often used bythose of ordinary skill to elucidate the function of particular genes.Antisense compounds are also used, for example, to distinguish betweenfunctions of various members of a biological pathway. Antisensemodulation has, therefore, been harnessed for research use.

[0025] For use in kits and diagnostics, the antisense compounds of thepresent invention, either alone or in combination with other antisensecompounds or therapeutics, can be used as tools in differential and/orcombinatorial analyses to elucidate expression patterns of a portion orthe entire complement of genes expressed within cells and tissues.

[0026] Expression patterns within cells or tissues treated with one ormore antisense compounds are compared to control cells or tissues nottreated with antisense compounds and the patterns produced are analyzedfor differential levels of gene expression as they pertain, for example,to disease association, signaling pathway, cellular localization,expression level, size, structure or function of the genes examined.These analyses can be performed on stimulated or unstimulated cells andin the presence or absence of other compounds which affect expressionpatterns.

[0027] Examples of methods of gene expression analysis known in the artinclude DNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000,480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serialanalysis of gene expression) (Madden, et al., Drug Discov. Today, 2000,5, 415-425), READS (restriction enzyme amplification of digested cDNAs)(Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (totalgene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci.U.S.A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, etal., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis,1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, etal., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000,80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal.Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41,203-208), subtractive cloning, differential display (DD) (Jurecic andBelmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomichybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31,286-96), FISH (fluorescent in situ hybridization) techniques (Going andGusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometrymethods (reviewed in (To, Comb. Chem. High Throughput Screen, 2000, 3,235-41).

[0028] The specificity and sensitivity of antisense is also harnessed bythose of skill in the art for therapeutic uses. Antisenseoligonucleotides have been employed as therapeutic moieties in thetreatment of disease states in animals and man. Antisenseoligonucleotide drugs, including ribozymes, have been safely andeffectively administered to humans and numerous clinical trials arepresently underway. It is thus established that oligonucleotides can beuseful therapeutic modalities that can be configured to be useful intreatment regimes for treatment of cells, tissues and animals,especially humans.

[0029] In the context of this invention, the term “oligonucleotide”refers to an oligomer or polymer of ribonucleic acid (RNA) ordeoxyribonucleic acid (DNA) or mimetics thereof. This term includesoligonucleotides composed of naturally-occurring nucleobases, sugars andcovalent internucleoside (backbone) linkages as well as oligonucleotideshaving non-naturally-occurring portions which function similarly. Suchmodified or substituted oligonucleotides are often preferred over nativeforms because of desirable properties such as, for example, enhancedcellular uptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases.

[0030] While antisense oligonucleotides are a preferred form ofantisense compound, the present invention comprehends other oligomericantisense compounds, including but not limited to oligonucleotidemimetics such as are described below. The antisense compounds inaccordance with this invention preferably comprise from about 8 to about50 nucleobases (i.e. from about 8 to about 50 linked nucleosides).Particularly preferred antisense compounds are antisenseoligonucleotides, even more preferably those comprising from about 12 toabout 30 nucleobases. Antisense compounds include ribozymes, externalguide sequence (EGS) oligonucleotides (oligozymes), and other shortcatalytic RNAs or catalytic oligonucleotides which hybridize to thetarget nucleic acid and modulate its expression.

[0031] As is known in the art, a nucleoside is a base-sugar combination.The base portion of the nucleoside is normally a heterocyclic base. Thetwo most common classes of such heterocyclic bases are the purines andthe pyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn the respective ends of this linear polymericstructure can be further joined to form a circular structure, however,open linear structures are generally preferred. Within theoligonucleotide structure, the phosphate groups are commonly referred toas forming the internucleoside backbone of the oligonucleotide. Thenormal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiesterlinkage.

[0032] Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides. Preferred modified oligonucleotidebackbones include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkyl-phosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiralphosphonates, phosphinates, phosphoramidates including 3′-aminophosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates,thiono-alkylphosphonates, thionoalkylphosphotriesters, selenophosphatesand boranophosphates having normal 3′-5′ linkages, 2′ -5′ linked analogsof these, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Preferred oligonucleotides having inverted polarity comprise a single 3′to 3′ linkage at the 3′-most internucleotide linkage i.e. a singleinverted nucleoside residue which may be abasic (the nucleobase ismissing or has a hydroxyl group in place thereof). Various salts, mixedsalts and free acid forms are also included.

[0033] Representative United States patents that teach the preparationof the above phosphorus-containing linkages include, but are not limitedto, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243;5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717;5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677;5,476,925; 5,519,126; 5,536,821; 5,541306; 5,550,111; 5,563,253;5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218;5,672,697 and 5,625,050, certain of which are commonly owned with thisapplication, and each of which is herein incorporated by reference.

[0034] Preferred modified oligonucleotide backbones that do not includea phosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

[0035] Representative United States patents that teach the preparationof the above oligonucleosides include, but are not limited to, U.S. Pat.Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain ofwhich are commonly owned with this application, and each of which isherein incorporated by reference.

[0036] In other preferred oligonucleotide mimetics, both the sugar andthe internucleoside linkage, i.e., the backbone, of the nucleotide unitsare replaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

[0037] Most preferred embodiments of the invention are oligonucleotideswith phosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [knownas a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃) —CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

[0038] Modified oligonucleotides may also contain one or moresubstituted sugar moieties. Preferred oligonucleotides comprise one ofthe following at the 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, orN-alkenyl; O—, S— or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred areO[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃,O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from1 to about 10. Other preferred oligonucleotides comprise one of thefollowing at the 2′ position: C₁ to C₁₀ lower alkyl, substituted loweralkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH,SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of anoligonucleotide, or a group for improving the pharmacodynamic propertiesof an oligonucleotide, and other substituents having similar properties.A preferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃,also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv.Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A furtherpreferred modification includes 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in exampleshereinbelow, and 2′-dimethylamino-ethoxyethoxy (also known in the art as2′-O-dimethylamino-ethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples hereinbelow.

[0039] A further prefered modification includes Locked Nucleic Acids(LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′ carbonatom of the sugar ring thereby forming a bicyclic sugar moiety. Thelinkage is preferably a methelyne (—CH₂—)_(n) group bridging the 2′oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs andpreparation thereof are described in WO 98/39352 and WO 99/14226.

[0040] Other preferred modifications include 2′-methoxy (2′-O—CH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl(2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be inthe arabino (up) position or ribo (down) position. A preferred2′-arabino modification is 2′-F. Similar modifications may also be madeat other positions on the oligonucleotide, particularly the 3′ positionof the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.Oligonucleotides may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar. Representative UnitedStates patents that teach the preparation of such modified sugarstructures include, but are not limited to, U.S. Pat. Nos. 4,981,957;5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633;5,792,747; and 5,700,920, certain of which are commonly owned with theinstant application, and each of which is herein incorporated byreference in its entirety.

[0041] Oligonucleotides may also include nucleobase (often referred toin the art simply as “base”) modifications or substitutions. As usedherein, “unmodified” or “natural” nucleobases include the purine basesadenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). Modified nucleobases include othersynthetic and natural nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl(—C≡C—CH₃) uracil and cytosine and other alkynyl derivatives ofpyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cyto-sines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modifiednucleobases include tricyclic pyrimidines such as phenoxazinecytidine(1H-pyrimido [5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′, 2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modifiednucleobases may also include those in which the purine or pyrimidinebase is replaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, pages858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosedby Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289-302, Crooke, S. T. and Lebleu, B.ed., CRC Press, 1993. Certain of these nucleobases are particularlyuseful for increasing the binding affinity of the oligomeric compoundsof the invention. These include 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including2-aminopropyl-adenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. andLebleu, B., eds., Antisense Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278) and are presently preferred basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

[0042] Representative United States patents that teach the preparationof certain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and5,681,941, certain of which are commonly owned with the instantapplication, and each of which is herein incorporated by reference, andU.S. Pat. No. 5,750,692, which is commonly owned with the instantapplication and also herein incorporated by reference.

[0043] Another modification of the oligonucleotides of the inventioninvolves chemically linking to the oligonucleotide one or more moietiesor conjugates which enhance the activity, cellular distribution orcellular uptake of the oligonucleotide. The compounds of the inventioncan include conjugate groups covalently bound to functional groups suchas primary or secondary hydroxyl groups. Conjugate groups of theinvention include inter-calators, reporter molecules, polyamines,polyamides, poly-ethylene glycols, polyethers, groups that enhance thepharmacodynamic properties of oligomers, and groups that enhance thepharmacokinetic properties of oligomers. Typical conjugates groupsinclude cholesterols, lipids, phospholipids, biotin, phenazine, folate,phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines,coumarins, and dyes. Groups that enhance the pharmaco-dynamicproperties, in the context of this invention, include groups thatimprove oligomer uptake, enhance oligomer resistance to degradation,and/or strengthen sequence-specific hybridization with RNA. Groups thatenhance the pharmacokinetic properties, in the context of thisinvention, include groups that improve oligomer uptake, distribution,metabolism or excretion. Representative conjugate groups are disclosedin International patent application PCT/US92/09196, filed Oct. 23, 1992the entire disclosure of which is incorporated herein by reference.Conjugate moieties include but are not limited to lipid moieties such asa cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA,1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem.Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharanet al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphaticchain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259,327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid,e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937). Oligonucleotides of the invention mayalso be conjugated to active drug substances, for example, aspirin,warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen,(S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoicacid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide,a diazepine, indo-methicin, a barbiturate., a cephalosporin, a sulfadrug, an antidiabetic, an antibacterial or an antibiotic.Oligonucleotide-drug conjugates and their preparation are described inU.S. patent application Ser. No. 09/334,130 (filed Jun. 15, 1999) whichis incorporated herein by reference in its entirety.

[0044] Representative United States patents that teach the preparationof such oligonucleotide conjugates include, but are not limited to, U.S.Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313;5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584;5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439;5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779;4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013;5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136;5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873;5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475;5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481;5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941,certain of which are commonly owned with the instant application, andeach of which is herein incorporated by reference.

[0045] It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide. The present invention alsoincludes antisense compounds which are chimeric compounds. “Chimeric”antisense compounds or “chimeras,” in the context of this invention, areantisense compounds, particularly oligonucleotides, which contain two ormore chemically distinct regions, each made up of at least one monomerunit, i.e., a nucleotide in the case of an oligonucleotide compound.These oligonucleotides typically contain at least one region wherein theoligonucleotide is modified so as to confer upon the oligonucleotideincreased resistance to nuclease degradation, increased cellular uptake,and/or increased binding affinity for the target nucleic acid. Anadditional region of the oligonucleotide may serve as a substrate forenzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way ofexample, RNase H is a cellular endonuclease which cleaves the RNA strandof an RNA:DNA duplex. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofoligonucleotide inhibition of gene expression. Consequently, comparableresults can often be obtained with shorter oligonucleotides whenchimeric oligonucleotides are used, compared to phosphorothioatedeoxyoligonucleotides hybridizing to the same target region. Cleavage ofthe RNA target can be routinely detected by gel electrophoresis and, ifnecessary, associated nucleic acid hybridization techniques known in theart.

[0046] Chimeric antisense compounds of the invention may be formed ascomposite structures of two or more oligonucleotides, modifiedoligonucleotides, oligonucleosides and/or oligonucleotide mimetics asdescribed above. Such compounds have also been referred to in the art ashybrids or gapmers. Representative United States patents that teach thepreparation of such hybrid structures include, but are not limited to,U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878;5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and5,700,922, certain of which are commonly owned with the instantapplication, and each of which is herein incorporated by reference inits entirety.

[0047] The antisense compounds used in accordance with this inventionmay be conveniently and routinely made through the well-known techniqueof solid phase synthesis. Equipment for such synthesis is sold byseveral vendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

[0048] The antisense compounds of the invention are synthesized in vitroand do not include antisense compositions of biological origin, orgenetic vector constructs designed to direct the in vivo synthesis ofantisense molecules.

[0049] The compounds of the invention may also be admixed, encapsulated,conjugated or otherwise associated with other molecules, moleculestructures or mixtures of compounds, as for example, liposomes, receptortargeted molecules, oral, rectal, topical or other formulations, forassisting in uptake, distribution and/or absorption. RepresentativeUnited States patents that teach the preparation of such uptake,distribution and/or absorption assisting formulations include, but arenot limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

[0050] The antisense compounds of the invention encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other compound which, upon administration to an animal including ahuman, is capable of providing (directly or indirectly) the biologicallyactive metabolite or residue thereof. Accordingly, for example, thedisclosure is also drawn to prodrugs and pharmaceutically acceptablesalts of the compounds of the invention, pharmaceutically acceptablesalts of such prodrugs, and other bioequivalents.

[0051] The term “prodrug” indicates a therapeutic agent that is preparedin an inactive form that is converted to an active form (i.e., drug)within the body or cells thereof by the action of endogenous enzymes orother chemicals and/or conditions. In particular, prodrug versions ofthe oligonucleotides of the invention are prepared as SATE[(S-acetyl-2-thioethyl) phosphate] derivatives according to the methodsdisclosed in WO-93/24510 to Gosselin et al., published Dec. 9, 1993 orin WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.

[0052] The term “pharmaceutically acceptable salts” refers tophysiologically and pharmaceutically acceptable salts of the compoundsof the invention: i.e., salts that retain the desired biologicalactivity of the parent compound and do not impart undesiredtoxicological effects thereto.

[0053] Pharmaceutically acceptable base addition salts are formed withmetals or amines, such as alkali and alkaline earth metals or organicamines. Examples of metals used as cations are sodium, potassium,magnesium, calcium, and the like. Examples of suitable amines areN,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine(see, for example, Berge et al., “Pharmaceutical Salts,” J. of PharmaSci., 1977, 66, 1-19). The base addition salts of said acidic compoundsare prepared by contacting the free acid form with a sufficient amountof the desired base to produce the salt in the conventional manner. Thefree acid form may be regenerated by contacting the salt form with anacid and isolating the free acid in the conventional manner. The freeacid forms differ from their respective salt forms somewhat in certainphysical properties such as solubility in polar solvents, but otherwisethe salts are equivalent to their respective free acid for purposes ofthe present invention. As used herein, a “pharmaceutical addition salt”includes a pharmaceutically acceptable salt of an acid form of one ofthe components of the compositions of the invention. These includeorganic or inorganic acid salts of the amines. Preferred acid salts arethe hydrochlorides, acetates, salicylates, nitrates and phosphates.Other suitable pharmaceutically acceptable salts are well known to thoseskilled in the art and include basic salts of a variety of inorganic andorganic acids, such as, for example, with inorganic acids, such as forexample hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoricacid; with organic carboxylic, sulfonic, sulfo or phospho acids orN-substituted sulfamic acids, for example acetic acid, propionic acid,glycolic acid, succinic acid, maleic acid, hydroxymaleic acid,methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid,oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, salicylic acid,4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid,embonic acid, nicotinic acid or isonicotinic acid; and with amino acids,such as the 20 alpha-amino acids involved in the synthesis of proteinsin nature, for example glutamic acid or aspartic acid, and also withphenylacetic acid, methanesulfonic acid, ethanesulfonic acid,2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,benzenesulfonic acid, 4-methylbenzenesulfoic acid,naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (withthe formation of cyclamates), or with other acid organic compounds, suchas ascorbic acid. Pharmaceutically acceptable salts of compounds mayalso be prepared with a pharmaceutically acceptable cation. Suitablepharmaceutically acceptable cations are well known to those skilled inthe art and include alkaline, alkaline earth, ammonium and quaternaryammonium cations. Carbonates or hydrogen carbonates are also possible.

[0054] For oligonucleotides, preferred examples of pharmaceuticallyacceptable salts include but are not limited to (a) salts formed withcations such as sodium, potassium, ammonium, magnesium, calcium,polyamines such as spermine and spermidine, etc.; (b) acid additionsalts formed with inorganic acids, for example hydrochloric acid,hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and thelike; (c) salts formed with organic acids such as, for example, aceticacid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaricacid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoicacid, tannic acid, palmitic acid, alginic acid, polyglutamic acid,naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine.

[0055] The antisense compounds of the present invention can be utilizedfor diagnostics, therapeutics, prophylaxis and as research reagents andkits. For therapeutics, an animal, preferably a human, suspected ofhaving a disease or disorder which can be treated by modulating theexpression of apolipoprotein(a) is treated by administering antisensecompounds in accordance with this invention. The compounds of theinvention can be utilized in pharmaceutical compositions by adding aneffective amount of an antisense compound to a suitable pharmaceuticallyacceptable diluent or carrier. Use of the antisense compounds andmethods of the invention may also be useful prophylactically, e.g., toprevent or delay infection, inflammation or tumor formation, forexample.

[0056] The antisense compounds of the invention are useful for researchand diagnostics, because these compounds hybridize to nucleic acidsencoding apolipoprotein(a), enabling sandwich and other assays to easilybe constructed to exploit this fact. Hybridization of the antisenseoligonucleotides of the invention with a nucleic acid encodingapolipoprotein(a) can be detected by means known in the art. Such meansmay include conjugation of an enzyme to the oligonucleotide,radiolabelling of the oligonucleotide or any other suitable detectionmeans. Kits using such detection means for detecting the level ofapolipoprotein(a) in a sample may also be prepared.

[0057] The present invention also includes pharmaceutical compositionsand formulations which include the antisense compounds of the invention.The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic and to mucousmembranes including vaginal and rectal delivery), pulmonary, e.g., byinhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal), oralor parenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g., intrathecal or intraventricular,administration. Oligonucleotides with at least one 2′-O-methoxyethylmodification are believed to be particularly useful for oraladministration.

[0058] Pharmaceutical compositions and formulations for topicaladministration may include transdermal patches, ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable. Coated condoms,gloves and the like may also be useful. Preferred topical formulationsinclude those in which the oligonucleotides of the invention are inadmixture with a topical delivery agent such as lipids, liposomes, fattyacids, fatty acid esters, steroids, chelating agents and surfactants.Preferred lipids and liposomes include neutral (e.g.dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl cholineDMPC, distearolyphosphatidyl choline) negative (e.g.dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). Oligonucleotides of the invention may beencapsulated within liposomes or may form complexes thereto, inparticular to cationic liposomes. Alternatively, oligonucleotides may becomplexed to lipids, in particular to cationic lipids. Preferred fattyacids and esters include but are not limited arachidonic acid, oleicacid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristicacid, palmitic acid, stearic acid, linoleic acid, linolenic acid,dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC₁₋₁₀ alkyl ester (e.g. isopropylmyristate IPM), monoglyceride,diglyceride or pharmaceutically acceptable salt thereof. Topicalformulations are described in detail in U.S. patent application Ser. No.09/315,298 filed on May 20, 1999 which is incorporated herein byreference in its entirety.

[0059] Compositions and formulations for oral administration includepowders or granules, microparticulates, nanoparticulates, suspensions orsolutions in water or non-aqueous media, capsules, gel capsules,sachets, tablets or minitablets. Thickeners, flavoring agents, diluents,emulsifiers, dispersing aids or binders may be desirable. Preferred oralformulations are those in which oligonucleotides of the invention areadministered in conjunction with one or more penetration enhancerssurfactants and chelators. Preferred surfactants include fatty acidsand/or esters or salts thereof, bile acids and/or salts thereof.Prefered bile acids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate, sodium glycodihydrofusidate. Preferedfatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g. sodium). Also prefered are combinations of penetrationenhancers, for example, fatty acids/salts in combination with bileacids/salts. A particularly prefered combination is the sodium salt oflauric acid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether.Oligonucleotides of the invention may be delivered orally in granularform including sprayed dried particles, or complexed to form micro ornanoparticles. Oligonucleotide complexing agents include poly-aminoacids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Particularly preferred complexing agentsinclude chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine,polyornithine, polyspermines, protamine, polyvinylpyridine,polythiodiethylamino-methylethylene P(TDAE), polyaminostyrene (e.g.p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor oligonucleotides and their preparation are described in detail inU.S. applications Ser. No. 08/886,829 (filed Jul. 1, 1997), Ser. No.09/108,673 (filed Jul. 1, 1998), Ser. No. 09/256,515 (filed Feb. 23,1999), Ser. No. 09/082,624 (filed May 21, 1998) and Ser. No. 09/315,298(filed May 20, 1999) each of which is incorporated herein by referencein their entirety.

[0060] Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

[0061] Pharmaceutical compositions of the present invention include, butare not limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

[0062] The pharmaceutical formulations of the present invention, whichmay conveniently be presented in unit dosage form, may be preparedaccording to conventional techniques well known in the pharmaceuticalindustry. Such techniques include the step of bringing into associationthe active ingredients with the pharmaceutical carrier(s) orexcipient(s). In general the formulations ate prepared by uniformly andintimately bringing into association the active ingredients with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product.

[0063] The compositions of the present invention may be formulated intoany of many possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

[0064] In one embodiment of the present invention the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product. The preparation of such compositions andformulations is generally known to those skilled in the pharmaceuticaland formulation arts and may be applied to the formulation of thecompositions of the present invention.

[0065] Emulsions

[0066] The compositions of the present invention may be prepared andformulated as emulsions. Emulsions are typically heterogenous systems ofone liquid dispersed in another in the form of droplets usuallyexceeding 0.1 μm in diameter. (Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 2, p. 335; Higuchi et al., in Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p.301). Emulsions are often biphasic systems comprising of two immiscibleliquid phases intimately mixed and dispersed with each other. Ingeneral, emulsions may be either water-in-oil (w/o) or of theoil-in-water (o/w) variety. When an aqueous phase is finely divided intoand dispersed as minute droplets into a bulk oily phase the resultingcomposition is called a water-in-oil (w/o) emulsion. Alternatively, whenan oily phase is finely divided into and dispersed as minute dropletsinto a bulk aqueous phase the resulting composition is called anoil-in-water (o/w) emulsion. Emulsions may contain additional componentsin addition to the dispersed phases and the active drug which may bepresent as a solution in either the aqueous phase, oily phase or itselfas a separate phase. Pharmaceutical excipients such as emulsifiers,stabilizers, dyes, and anti-oxidants may also be present in emulsions asneeded. Pharmaceutical emulsions may also be multiple emulsions that arecomprised of more than two phases such as, for example, in the case ofoil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.Such complex formulations often provide certain advantages that simplebinary emulsions do not. Multiple emulsions in which individual oildroplets of an o/w emulsion enclose small water droplets constitute aw/o/w emulsion. Likewise a system of oil droplets enclosed in globulesof water stabilized in an oily continuous provides an o/w/o emulsion.

[0067] Emulsions are characterized by little or no thermodynamicstability. Often, the dispersed or discontinuous phase of the emulsionis well dispersed into the external or continuous phase and maintainedin this form through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

[0068] Synthetic surfactants, also known as surface active agents, havefound wide applicability in the formulation of emulsions and have beenreviewed in the literature (Rieger, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York,N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic andcomprise a hydrophilic and a hydrophobic portion. The ratio of thehydrophilic to the hydrophobic nature of the surfactant has been termedthe hydrophile/lipophile balance (HLB) and is a valuable tool incategorizing and selecting surfactants in the preparation offormulations. Surfactants may be classified into different classes basedon the nature of the hydrophilic group: nonionic, anionic, cationic andamphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 285).

[0069] Naturally occurring emulsifiers used in emulsion formulationsinclude lanolin, beeswax, phosphatides, lecithin and acacia. Absorptionbases possess hydrophilic properties such that they can soak up water toform w/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

[0070] A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

[0071] Hydrophilic colloids or hydrocolloids include naturally occurringgums and synthetic polymers such as polysaccharides (for example,acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, andtragacanth), cellulose derivatives (for example, carboxymethylcelluloseand carboxypropylcellulose), and synthetic polymers (for example,carbomers, cellulose ethers, and carboxyvinyl polymers). These disperseor swell in water to form colloidal solutions that stabilize emulsionsby forming strong interfacial films around the dispersed-phase dropletsand by increasing the viscosity of the external phase.

[0072] Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used may be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

[0073] The application of emulsion formulations via dermatological, oraland parenteral routes and methods for their manufacture have beenreviewed in the literature (Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 199). Emulsion formulations for oral deliveryhave been very widely used because of reasons of ease of formulation,efficacy from an absorption and bioavailability standpoint. (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil baselaxatives, oil-soluble vitamins and high fat nutritive preparations areamong the materials that have commonly been administered orally as o/wemulsions.

[0074] In one embodiment of the present invention, the compositions ofoligonucleotides and nucleic acids are formulated as microemulsions. Amicroemulsion may be defined as a system of water, oil and amphiphilewhich is a single optically isotropic and thermodynamically stableliquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 245). Typically microemulsions are systems that areprepared by first dispersing an oil in an aqueous surfactant solutionand then adding a sufficient amount of a fourth component, generally anintermediate chain-length alcohol to form a transparent system.Therefore, microemulsions have also been described as thermodynamicallystable, isotropically clear dispersions of two immiscible liquids thatare stabilized by interfacial films of surface-active molecules (Leungand Shah, in: Controlled Release of Drugs: Polymers and AggregateSystems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages185-215). Microemulsions commonly are prepared via a combination ofthree to five components that include oil, water, surfactant,cosurfactant and electrolyte. Whether the microemulsion is of thewater-in-oil (w/o) or an oil-in-water (o/w) type is dependent on theproperties of the oil and surfactant used and on the structure andgeometric packing of the-polar heads and hydrocarbon tails of thesurfactant molecules (Schott, in Remington's Pharmaceutical Sciences,Mack Publishing Co., Easton, Pa., 1985, p. 271).

[0075] The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared toconventional emulsions, microemulsions offer the advantage ofsolubilizing water-insoluble drugs in a formulation of thermodynamicallystable droplets that are formed spontaneously.

[0076] Surfactants used in the preparation of microemulsions include,but are not limited to, ionic surfactants, non-ionic surfactants, Brij96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions may, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase may typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase may include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

[0077] Microemulsions are particularly of interest from the standpointof drug solubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm.Sci., 1996, 85, 138-143). Often microemulsions may form spontaneouslywhen their components are brought together at ambient temperature. Thismay be particularly advantageous when formulating thermolabile drugs,peptides or oligonucleotides. Microemulsions have also been effective inthe transdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of oligonucleotides and nucleic acidsfrom the gastrointestinal tract, as well as improve the local cellularuptake of oligonucleotides and nucleic acids within the gastrointestinaltract, vagina, buccal cavity and other areas of administration.

[0078] Microemulsions of the present invention may also containadditional components and additives such as sorbitan monostearate (Grill3), Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the oligonucleotides andnucleic acids of the present invention. Penetration enhancers used inthe microemulsions of the present invention may be classified asbelonging to one of five broad categories—surfactants, fatty acids, bilesalts, chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

[0079] Liposomes

[0080] There are many organized surfactant structures besidesmicroemulsions that have been studied and used for the formulation ofdrugs. These include monolayers, micelles, bilayers and vesicles.Vesicles, such as liposomes, have attracted great interest because oftheir specificity and the duration of action they offer from thestandpoint of drug delivery. As used in the present invention, the term“liposome” means a vesicle composed of amphiphilic lipids arranged in aspherical bilayer or bilayers.

[0081] Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo.

[0082] In order to cross intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome which is highly deformable and able to passthrough such fine pores.

[0083] Further advantages of liposomes include; liposomes obtained fromnatural phospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245). Important considerations in thepreparation of liposome formulations are the lipid surface charge,vesicle size and the aqueous volume of the liposomes.

[0084] Liposomes are useful for the transfer and delivery of activeingredients to the site of action. Because the liposomal membrane isstructurally similar to biological membranes, when liposomes are appliedto a tissue, the liposomes start to merge with the cellular membranes.As the merging of the liposome and cell progresses, the liposomalcontents are emptied into the cell where the active agent may act.

[0085] Liposomal formulations have been the focus of extensiveinvestigation as the mode of delivery for many drugs. There is growingevidence that for topical administration, liposomes present severaladvantages over other formulations. Such advantages include reducedside-effects related to high systemic absorption of the administereddrug, increased accumulation of the administered drug at the desiredtarget, and the ability to administer a wide variety of drugs, bothhydrophilic and hydrophobic, into the skin.

[0086] Several reports have detailed the ability of liposomes to deliveragents including high-molecular weight DNA into the skin. Compoundsincluding analgesics, antibodies, hormones and high-molecular weightDNAs have been administered to the skin. The majority of applicationsresulted in the targeting of the upper epidermis.

[0087] Liposomes fall into two broad classes. Cationic liposomes arepositively charged liposomes which interact with the negatively chargedDNA molecules to form a stable complex. The positively chargedDNA/liposome complex binds to the negatively charged cell surface and isinternalized in an endosome. Due to the acidic pH within the endosome,the liposomes are ruptured, releasing their contents into the cellcytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147,980-985).

[0088] Liposomes which are pH-sensitive or negatively-charged, entrapDNA rather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs.Nevertheless, some DNA is entrapped within the aqueous interior of theseliposomes. pH-sensitive liposomes have been used to deliver DNA encodingthe thymidine kinase gene to cell monolayers in culture. Expression ofthe exogenous gene was detected in the target cells (Zhou et al.,Journal of Controlled Release, 1992, 19, 269-274).

[0089] One major type of liposomal composition includes phospholipidsother than naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

[0090] Several studies have assessed the topical delivery of liposomaldrug formulations to the skin. Application of liposomes containinginterferon to guinea pig skin resulted in a reduction of skin herpessores while delivery of interferon via other means (e.g. as a solutionor as an emulsion) were ineffective (Weiner et al., Journal of DrugTargeting, 1992, 2, 405-410). Further, an additional study tested theefficacy of interferon administered as part of a liposomal formulationto the administration of interferon using an aqueous system, andconcluded that the liposomal formulation was superior to aqueousadministration (du Plessis et al., Antiviral Research, 1992, 18,259-265).

[0091] Non-ionic liposomal systems have also been examined to determinetheir utility in the delivery of drugs to the skin, in particularsystems comprising non-ionic surfactant and cholesterol. Non-ionicliposomal formulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporin-A into different layers ofthe skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4, 6, 466).

[0092] Liposomes also include “sterically stabilized” liposomes, a termwhich, as used herein, refers to liposomes comprising one or morespecialized lipids that, when incorporated into liposomes, result inenhanced circulation lifetimes relative to liposomes lacking suchspecialized lipids. Examples of sterically stabilized liposomes arethose in which part of the vesicle-forming lipid portion of the liposome(A) comprises one or more glycolipids, such as monosialogangliosideG_(M1), or (B) is derivatized with one or more hydrophilic polymers,such as a polyethylene glycol (PEG) moiety. While not wishing to bebound by any particular theory, it is thought in the art that, at leastfor sterically stabilized liposomes containing gangliosides,sphingomyelin, or PEG-derivatized lipids, the enhanced circulationhalf-life of these sterically stabilized liposomes derives from areduced uptake into cells of the reticuloendothelial system (RES) (Allenet al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993,53, 3765). Various liposomes comprising one or more glycolipids areknown in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987,507, 64) reported the ability of monosialoganglioside G_(M1),galactocerebroside sulfate and phosphatidylinositol to improve bloodhalf-lives of liposomes. These findings were expounded upon by Gabizonet al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No.4,837,028 and WO 88/04924, both to Allen et al., disclose liposomescomprising (1) sphingomyelin and (2) the ganglioside G_(M1) or agalactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.)discloses liposomes comprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al.).

[0093] Many liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778)described liposomes comprising a nonionic detergent, 2C₁₂15G, thatcontains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) notedthat hydrophilic coating of polystyrene particles with polymeric glycolsresults in significantly enhanced blood half-lives. Syntheticphospholipids modified by the attachment of carboxylic groups ofpolyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235)described experiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta, 1990, 1029, 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. EP 0 445 131 B1and WO 90/04384 to Fisher. Liposome compositions containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) andMartin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496813 B1). Liposomes comprising a number of other lipid-polymer conjugatesare disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martinet al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprisingPEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.).U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948(Tagawa et al.) describe PEG-containing liposomes that can be furtherderivatized with functional moieties on their surfaces.

[0094] A limited number of liposomes comprising nucleic acids are knownin the art. WO 96/40062 to Thierry et al. discloses methods forencapsulating high molecular weight nucleic acids in liposomes. U.S.Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomesand asserts that the contents of such liposomes may include an antisenseRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methodsof encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Loveet al. discloses liposomes comprising antisense oligonucleotidestargeted to the raf gene.

[0095] Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes may be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g. they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

[0096] Surfactants find wide application in formulations such asemulsions (including microemulsions) and liposomes. The most common wayof classifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

[0097] If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

[0098] If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

[0099] If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

[0100] If the surfactant molecule has the ability to carry either apositive or negative charge, the surfactant is classified as amphoteric.Amphoteric surfactants include acrylic acid derivatives, substitutedalkylamides, N-alkylbetaines and phosphatides.

[0101] The use of surfactants in drug products, formulations and inemulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms,Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

[0102] Penetration Enhancers

[0103] In one embodiment, the present invention employs variouspenetration enhancers to effect the efficient delivery of nucleic acids,particularly oligonucleotides, to the skin of animals. Most drugs arepresent in solution in both ionized and nonionized forms. However,usually only lipid soluble or lipophilic drugs readily cross cellmembranes. It has been discovered that even non-lipophilic drugs maycross cell membranes if the membrane to be crossed is treated with apenetration enhancer. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs.

[0104] Penetration enhancers may be classified as belonging to one offive broad categories, i.e., surfactants, fatty acids, bile salts,chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92). Eachof the above mentioned classes of penetration enhancers are describedbelow in greater detail.

[0105] Surfactants: In connection with the present invention,surfactants (or “surface-active agents”) are chemical entities which,when dissolved in an aqueous solution, reduce the surface tension of thesolution or the interfacial tension between the aqueous solution andanother liquid, with the result that absorption of oligonucleotidesthrough the mucosa is enhanced. In addition to bile salts and fattyacids, these penetration enhancers include, for example, sodium laurylsulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetylether) (Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, p.92); and perfluorochemical emulsions, such as FC-43.Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

[0106] Fatty acids: Various fatty acids and their derivatives which actas penetration. enhancers include, for example, oleic acid, lauric acid,capric acid (n-decanoic acid), myristic acid, palmitic acid, stearicacid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C₁₋₁₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92;Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

[0107] Bile salts: The physiological role of bile includes thefacilitation of dispersion and absorption of lipids and fat-solublevitamins (Brunton, Chapter 38 in: Goodman & Gilman's The PharmacologicalBasis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, NewYork, 1996, pp. 934-935). Various natural bile salts, and theirsynthetic derivatives, act as penetration enhancers. Thus the term “bilesalts” includes any of the naturally occurring components of bile aswell as any of their synthetic derivatives. The bile salts of theinvention include, for example, cholic acid (or its pharmaceuticallyacceptable sodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium-taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee etal., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18thEd., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages782-783; Muranishi, Critical Reviews in Therapeutic Drug CarrierSystems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992,263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

[0108] Chelating Agents: Chelating agents, as used in connection withthe present invention, can be defined as compounds that remove metallicions from solution by forming complexes therewith, with the result thatabsorption of oligonucleotides through the mucosa is enhanced. Withregards to their use as penetration enhancers in the present invention,chelating agents have the added advantage of also serving as DNaseinhibitors, as most characterized DNA nucleases require a divalent metalion for catalysis and are thus inhibited by chelating agents (Jarrett,J. Chromatogr., 1993, 618, 315-339). Chelating agents of the inventioninclude but are not limited to disodium ethylenediaminetetraacetate(EDTA), citric acid, salicylates (e.g., sodium salicylate,5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen,laureth-9 and N-amino acyl derivatives of beta-diketones (enamines) (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems,1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

[0109] Non-chelating non-surfactants: As used herein, non-chelatingnon-surfactant penetration enhancing compounds can be defined ascompounds that demonstrate insignificant activity as chelating agents oras surfactants but that nonetheless enhance absorption ofoligonucleotides through the alimentary mucosa (Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This classof penetration enhancers include, for example, unsaturated cyclic ureas,1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92);and non-steroidal anti-inflammatory agents such as diclofenac sodium,indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol.,1987, 39, 621-626).

[0110] Agents that enhance uptake of oligonucleotides at the cellularlevel may also be added to the pharmaceutical and other compositions ofthe present invention. For example, cationic lipids, such as lipofectin(Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives,and polycationic molecules, such as polylysine (Lollo et al., PCTapplication WO 97/30731), are also known to enhance the cellular uptakeof oligonucleotides.

[0111] Other agents may be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

[0112] Carriers

[0113] Certain compositions of the present invention also incorporatecarrier compounds in the formulation. As used herein, “carrier compound”or “carrier” can refer to a nucleic acid, or analog thereof, which isinert (i.e., does not possess biological activity per se) but isrecognized as a nucleic acid by in vivo processes that reduce thebioavailability of a nucleic acid having biological activity by, forexample, degrading the biologically active nucleic acid or promoting itsremoval from circulation. The coadministration of a nucleic acid and acarrier compound, typically with an excess of the latter substance, canresult in a substantial reduction of the amount of nucleic acidrecovered in the liver, kidney or other extracirculatory reservoirs,presumably due to competition between the carrier compound and thenucleic acid for a common receptor. For example, the recovery of apartially phosphorothioate oligonucleotide in hepatic tissue can bereduced when it is coadministered with polyinosinic acid, dextransulfate, polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al., Antisense Res.Dev., 1995, 5, 115-121; Takakura et al., Antisense & Nucl. Acid DrugDev., 1996, 6, 177-183).

[0114] Excipients

[0115] In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc.).

[0116] Pharmaceutically acceptable organic or inorganic excipientsuitable for non-parenteral administration which do not deleteriouslyreact with nucleic acids can also be used to formulate the compositionsof the present invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

[0117] Formulations for topical administration of nucleic acids mayinclude sterile and non-sterile aqueous solutions, non-aqueous solutionsin common solvents such as alcohols, or solutions of the nucleic acidsin liquid or solid oil bases. The solutions may also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

[0118] Suitable pharmaceutically acceptable excipients include, but arenot limited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

[0119] Other Components

[0120] The compositions of the present invention may additionallycontain other adjunct components conventionally found in pharmaceuticalcompositions, at their art-established usage levels. Thus, for example,the compositions may contain additional, compatible,pharmaceutically-active materials such as, for example, antipruritics,astringents, local anesthetics or anti-inflammatory agents, or maycontain additional materials useful in physically formulating variousdosage forms of the compositions of the present invention, such as dyes,flavoring agents, preservatives, antioxidants, opacifiers, thickeningagents and stabilizers. However, such materials, when added, should notunduly interfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

[0121] Aqueous suspensions may contain substances which increase theviscosity of the suspension including, for example, sodiumcarboxymethylcellulose, sorbitol and/or dextran. The suspension may alsocontain stabilizers.

[0122] Certain embodiments of the invention provide pharmaceuticalcompositions containing (a) one or more antisense compounds and (b) oneor more other chemotherapeutic agents which function by a non-antisensemechanism. Examples of such chemotherapeutic agents include but are notlimited to daunorubicin, daunomycin, dactinomycin, doxorubicin,epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide,cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C,actinomycin D, mithramycin, prednisone, hydroxyprogesterone,testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine,pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil,methylcyclohexylnitrosurea, nitrogen mustards, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol(DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15thEd. 1987, pp. 1206-1228, Berkow et al., eds., Rahway, N.J. When usedwith the compounds of the invention, such chemotherapeutic agents may beused individually (e.g., 5-FU and oligonucleotide), sequentially (e.g.,5-FU and oligonucleotide for a period of time followed by MTX andoligonucleotide), or in combination with one or more other suchchemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU,radiotherapy and oligonucleotide). Anti-inflammatory drugs, includingbut not limited to nonsteroidal anti-inflammatory drugs andcorticosteroids, and antiviral drugs, including but not limited toribivirin, vidarabine, acyclovir and ganciclovir, may also be combinedin compositions of the invention. See, generally, The Merck Manual ofDiagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway,N.J., pages 2499-2506 and 46-49, respectively). Other non-antisensechemotherapeutic agents are also within the scope of this invention. Twoor more combined compounds may be used together or sequentially.

[0123] In another related embodiment, compositions of the invention maycontain one or more antisense compounds, particularly oligonucleotides,targeted to a first nucleic acid and one or more additional antisensecompounds targeted to a second nucleic acid target. Numerous examples ofantisense compounds are known in the art. Two or more combined compoundsmay be used together or sequentially.

[0124] The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient.Persons of ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC₅₀s found to be effective in in vitroand in vivo animal models. In general, dosage is from 0.01 ug to 100 gper kg of body weight, and may be given once or more daily, weekly,monthly or yearly, or even once every 2 to 20 years. Persons of ordinaryskill in the art can easily estimate repetition rates for dosing basedon measured residence times and concentrations of the drug in bodilyfluids or tissues. Following successful treatment, it may be desirableto have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 ug to 100 g per kgof body weight, once or more daily, to once every 20 years.

[0125] While the present invention has been described with specificityin accordance with certain of its preferred embodiments, the followingexamples serve only to illustrate the invention and are not intended tolimit the same.

EXAMPLES Example 1 Nucleoside Phosphoramidites for OligonucleotideSynthesis Deoxy and 2′-alkoxy amidites

[0126] 2′-Deoxy and 2′-methoxy beta-cyanoethyldiisopropylphosphoramidites were purchased from commercial sources (e.g. Chemgenes,Needham Mass. or Glen Research, Inc. Sterling Va.). Other 2′-O-alkoxysubstituted nucleoside amidites are prepared as described in U.S. Pat.No. 5,506,351, herein incorporated by reference. For oligonucleotidessynthesized using 2′-alkoxy amidites, the standard cycle for unmodifiedoligonucleotides was utilized, except the wait step after pulse deliveryof tetrazole and base was increased to 360 seconds.

[0127] Oligonucleotides containing 5-methyl-2′-deoxycytidine (5-Me-C)nucleotides were synthesized according to published methods (Sanghvi,et. al., Nucleic Acids Research, 1993, 21, 3197-3203) using commerciallyavailable phosphoramidites (Glen Research, Sterling Va. or ChemGenes,Needham Mass.).

2′-Fluoro amidites 2′-Fluorodeoxyadenosine amidites

[0128] 2′-fluoro oligonucleotides were synthesized as describedpreviously (Kawasaki, et. al., J. Med. Chem., 1993, 36, 831-841) andU.S. Pat. No. 5,670,633, herein incorporated by reference. Briefly, theprotected nucleoside N6-benzoyl-2′-deoxy-2′-fluoroadenosine wassynthesized utilizing commercially available9-beta-D-arabinofuranosyladenine as starting material and by modifyingliterature procedures whereby the 2′-alpha-fluoro atom is introduced bya S_(N)2-displacement of a 2′-beta-trityl group. ThusN6-benzoyl-9-beta-D-arabinofuranosyladenine was selectively protected inmoderate yield as the 3′,5′-ditetrahydropyranyl (THP) intermediate.Deprotection of the THP and N6-benzoyl groups was accomplished usingstandard methodologies and standard methods were used to obtain the5′-dimethoxytrityl-(DMT) and 5′-DMT-3′-phosphoramidite intermediates.

2′-Fluorodeoxyguanosine

[0129] The synthesis of 2′-deoxy-2′-fluoroguanosine was accomplishedusing tetraisopropyldisiloxanyl (TPDS) protected9-beta-D-arabinofuranosylguanine as starting material, and conversion tothe intermediate diisobutyryl-arabinofuranosylguanosine. Deprotection ofthe TPDS group was followed by protection of the hydroxyl group with THPto give diisobutyryl di-THP protected arabinofuranosylguanine. SelectiveO-deacylation and triflation was followed by treatment of the crudeproduct with fluoride, then deprotection of the THP groups. Standardmethodologies were used to obtain the 5′-DMT- and5′-DMT-3′-phosphoramidites.

2′-Fluorouridine

[0130] Synthesis of 2′-deoxy-2′-fluorouridine was accomplished by themodification of a literature procedure in which2,2′-anhydro-1-beta-D-arabinofuranosyluracil was treated with 70%hydrogen fluoride-pyridine. Standard procedures were used to obtain the5′-DMT and 5′-DMT-3′phosphoramidites.

2′-Fluorodeoxycytidine

[0131] 2′-deoxy-2′-fluorocytidine was synthesized via amination of2′-deoxy-2′-fluorouridine, followed by selective protection to giveN4-benzoyl-2′-deoxy-2′-fluorocytidine. Standard procedures were used toobtain the 5′-DMT and 5′-DMT-3′phosphoramidites.

2′-O-(2-Methoxyethyl) modified amidites

[0132] 2′-O-Methoxyethyl-substituted nucleoside amidites are prepared asfollows, or alternatively, as per the methods of Martin, P., HelveticaChimica Acta, 1995, 78, 486-504.

2,2′-Anhydro[1-(beta-D-arabinofuranosyl)-5-methyluridine]

[0133] 5-Methyluridine (ribosylthymine, commercially available throughYamasa, Choshi, Japan) (72.0 g, 0.279 M), diphenylcarbonate (90.0 g,0.420 M) and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300mL). The mixture was heated to reflux, with stirring, allowing theevolved carbon dioxide gas to be released in a controlled manner. After1 hour, the slightly darkened solution was concentrated under reducedpressure. The resulting syrup was poured into diethylether (2.5 L), withstirring. The product formed a gum. The ether was decanted and theresidue was dissolved in a minimum amount of methanol (ca. 400 mL). Thesolution was poured into fresh ether (2.5 L) to yield a stiff gum. Theether was decanted and the gum was dried in a vacuum oven (60° C. at 1mm Hg for 24 h) to give a solid that was crushed to a light tan powder(57 g, 85% crude yield). The NMR spectrum was consistent with thestructure, contaminated with phenol as its sodium salt (ca. 5%). Thematerial was used as is for further reactions (or it can be purifiedfurther by column chromatography using a gradient of methanol in ethylacetate (10-25%) to give a white solid, mp 222-4° C.)

2′-O-Methoxyethyl-5-methyluridine

[0134] 2,2′-Anhydro-5-methyluridine (195 g, 0.81 M),tris(2-methoxyethyl)borate (231 g, 0.98 M) and 2-methoxyethanol (1.2 L)were added to a 2 L stainless steel pressure vessel and placed in apre-heated oil bath at 160° C. After heating for 48 hours at 155-160°C., the vessel was opened and the solution evaporated to dryness andtriturated with MeOH (200 mL). The residue was suspended in hot acetone(1 L). The insoluble salts were filtered, washed with acetone (150 mL)and the filtrate evaporated. The residue (280 g) was dissolved in CH₃CN(600 mL) and evaporated. A silica gel column (3 kg) was packed inCH₂Cl₂/acetone/MeOH (20:5:3) containing 0.5% Et₃NH. The residue wasdissolved in CH₂Cl₂ (250 mL) and adsorbed onto silica (150 g) prior toloading onto the column. The product was eluted with the packing solventto give 160 g (63%) of product. Additional material was obtained byreworking impure fractions.

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

[0135] 2′-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) wasco-evaporated with pyridine (250 mL) and the dried residue dissolved inpyridine (1.3 L). A first aliquot of dimethoxytrityl chloride (94.3 g,0.278 M) was added and the mixture stirred at room temperature for onehour. A second aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) wasadded and the reaction stirred for an additional one hour. Methanol (170mL) was then added to stop the reaction. HPLC showed the presence ofapproximately 70% product. The solvent was evaporated and trituratedwith CH₃CN (200 mL). The residue was dissolved in CHCl₃ (1.5 L) andextracted with 2×500 mL of saturated NaHCO₃ and 2×500 mL of saturatedNaCl. The organic phase was dried over Na₂SO₄, filtered and evaporated.275 g of residue was obtained. The residue was purified on a 3.5 kgsilica gel column, packed and eluted with EtOAc/hexane/acetone (5:5:1)containing 0.5% Et₃NH. The pure fractions were evaporated to give 164 gof product. Approximately 20 g additional was obtained from the impurefractions to give a total yield of 183 g (57%).

3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

[0136] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (106 g,0.167 M), DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL ofDMF and 188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M)were combined and stirred at room temperature for 24 hours. The reactionwas monitored by TLC by first quenching the TLC sample with the additionof MeOH. Upon completion of the reaction, as judged by TLC, MeOH (50 mL)was added and the mixture evaporated at 35° C. The residue was dissolvedin CHCl₃ (800 mL) and extracted with 2×200 mL of saturated sodiumbicarbonate and 2×200 mL of saturated NaCl. The water layers were backextracted with 200 mL of CHCl₃. The combined organics were dried withsodium sulfate and evaporated to give 122 g of residue (approx. 90%product). The residue was purified on a 3.5 kg silica gel column andeluted using EtOAc/hexane(4:1). Pure product fractions were evaporatedto yield 96 g (84%). An additional 1.5 g was recovered from laterfractions.

3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine

[0137] A first solution was prepared by dissolving3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (96g, 0.144 M) in CH₃CN (700 mL) and set aside. Triethylamine (189 mL, 1.44M) was added to a solution of triazole (90 g, 1.3 M) in CH₃CN (1 L),cooled to −5° C. and stirred for 0.5 h using an overhead stirrer. POCl₃was added dropwise, over a 30 minute period, to the stirred solutionmaintained at 0-10° C., and the resulting mixture stirred for anadditional 2hours. The first solution was added dropwise, over a 45minute period, to the latter solution. The resulting reaction mixturewas stored overnight in a cold room. Salts were filtered from thereaction mixture and the solution was evaporated. The residue wasdissolved in EtOAc (1 L) and the insoluble solids were removed byfiltration. The filtrate was washed with 1×300 mL of NaHCO₃ and 2×300 mLof saturated NaCl, dried over sodium sulfate and evaporated. The residuewas triturated with EtOAc to give the title compound.

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

[0138] A solution of3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine(103 g, 0.141 M) in dioxane (500 mL) and NH₄OH (30 mL) was stirred atroom temperature for 2 hours. The dioxane solution was evaporated andthe residue azeotroped with MeOH (2×200 mL). The residue was dissolvedin MeOH (300 mL) and transferred to a 2 liter stainless steel pressurevessel. MeOH (400 mL) saturated with NH₃ gas was added and the vesselheated to 100° C. for 2 hours (TLC showed complete conversion). Thevessel contents were evaporated to dryness and the residue was dissolvedin EtOAc (500 mL) and washed once with saturated NaCl (200 mL). Theorganics were dried over sodium sulfate and the solvent was evaporatedto give 85 g (95%) of the title compound.

N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

[0139] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyl-cytidine (85 g,0.134 M) was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g,0.165 M) was added with stirring. After stirring for 3 hours, TLC showedthe reaction to be approximately 95% complete. The solvent wasevaporated and the residue azeotroped with MeOH (200 mL). The residuewas dissolved in CHCl₃ (700 mL) and extracted with saturated NaHCO₃(2×300 mL) and saturated NaCl (2×300 mL), dried over MgSO₄ andevaporated to give a residue (96 g). The residue was chromatographed ona 1.5 kg silica column using EtOAc/hexane (1:1) containing 0.5% Et₃NH asthe eluting solvent. The pure product fractions were evaporated to give90 g (90%) of the title compound.

N4-Benzoyl-2′-N-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine-3′-amidite

[0140]N4-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (74g, 0.10 M) was dissolved in CH₂Cl₂ (1 L). Tetrazole diisopropylamine(7.1 g) and 2-cyanoethoxy-tetra-(isopropyl)phosphite (40.5 mL, 0.123 M)were added with stirring, under a nitrogen atmosphere. The resultingmixture was stirred for 20 hours at room temperature (TLC showed thereaction to be 95% complete). The reaction mixture was extracted withsaturated NaHCO₃ (1×300 mL) and saturated NaCl (3×300 mL). The aqueouswashes were back-extracted with CH₂Cl₂ (300 mL), and the extracts werecombined, dried over MgSO₄ and concentrated. The residue obtained waschromatographed on a 1.5 kg silica column using EtOAc/hexane (3:1) asthe eluting solvent. The pure fractions were combined to give 90.6 g(87%) of the title compound.

2′-O-(Aminooxyethyl) nucleoside amidites and2′-O-(dimethylaminooxyethyl) nucleoside amidites2′-(Dimethylaminooxyethoxy) nucleoside amidites

[0141] 2′-(Dimethylaminooxyethoxy) nucleoside amidites (also known inthe art as 2′-O-(dimethylaminooxyethyl) nucleoside amidites) areprepared as described in the following paragraphs. Adenosine, cytidineand guanosine nucleoside amidites are prepared similarly to thethymidine (5-methyluridine) except the exocyclic amines are protectedwith a benzoyl moiety in the case of adenosine and cytidine and withisobutyryl in the case of guanosine.

5-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine

[0142] O²-2′-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy,100.0 g, 0.416 mmol), dimethylaminopyridine (0.66 g, 0.013 eq, 0.0054mmol) were dissolved in dry pyridine (500 ml) at ambient temperatureunder an argon atmosphere and with mechanical stirring.tert-Butyldiphenylchlorosilane (125.8 g, 119.0 mL, 1.1 eq, 0.458 mmol)was added in one portion. The reaction was stirred for 16 h at ambienttemperature. TLC (Rf 0.22, ethyl acetate) indicated a complete reaction.The solution was concentrated under reduced pressure to a thick oil.This was partitioned between dichloromethane (1 L) and saturated sodiumbicarbonate (2×1 L) and brine (1 L). The organic layer was dried oversodium sulfate and concentrated under reduced pressure to a thick oil.The oil was dissolved in a 1:1 mixture of ethyl acetate and ethyl ether(600 mL) and the solution was cooled to −10° C. The resultingcrystalline product was collected by filtration, washed with ethyl ether(3×200 mL) and dried (40° C., 1 mm Hg, 24 h) to 149 g (74.8%) of whitesolid. TLC and NMR were consistent with pure product.

5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine

[0143] In a 2 L stainless steel, unstirred pressure reactor was addedborane in tetrahydrofuran (1.0 M, 2.0 eq, 622 mL). In the fume hood andwith manual stirring, ethylene glycol (350 mL, excess) was addedcautiously at first until the evolution of hydrogen gas subsided.5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine (149 g, 0.311mol) and sodium bicarbonate (0.074 g, 0.003 eq) were added with manualstirring. The reactor was sealed and heated in an oil bath until aninternal temperature of 160° C. was reached and then maintained for 16 h(pressure<100 psig). The reaction vessel was cooled to ambient andopened. TLC (Rf 0.67 for desired product and Rf 0.82 for ara-T sideproduct, ethyl acetate) indicated about 70% conversion to the product.In order to avoid additional side product formation, the reaction wasstopped, concentrated under reduced pressure (10 to 1 mm Hg) in a warmwater bath (40-100° C.) with the more extreme conditions used to removethe ethylene glycol. [Alternatively, once the low boiling solvent isgone, the remaining solution can be partitioned between ethyl acetateand water. The product will be in the organic phase.] The residue waspurified by column chromatography (2 kg silica gel, ethylacetate-hexanes gradient 1:1 to 4:1). The appropriate fractions werecombined, stripped and dried to product as a white crisp foam (84 g,50%), contaminated starting material (17.4 g) and pure reusable startingmaterial 20 g. The yield based on starting material less pure recoveredstarting material was 58%. TLC and NMR were consistent with 99% pureproduct.

2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine

[0144] 5′-O-tert-Butyldiphenylsilyl-2-O-(2-hydroxyethyl)-5-methyluridine(20 g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g, 44.36mmol) and N-hydroxyphthalimide (7.24 g, 44.36 mmol). It was then driedover P₂O₅ under high vacuum for two days at 40° C. The reaction mixturewas flushed with argon and dry THF (369.8 mL, Aldrich, sure seal bottle)was added to get a clear solution. Diethyl-azodicarboxylate (6.98 mL,44.36 mmol) was added dropwise to the reaction mixture. The rate ofaddition is maintained such that resulting deep red coloration is justdischarged before adding the next drop. After the addition was complete,the reaction was stirred for 4 hrs. By that time TLC showed thecompletion of the reaction (ethylacetate:hexane, 60:40). The solvent wasevaporated in vacuum. Residue obtained was placed on a flash column andeluted with ethyl acetate:hexane (60:40), to get2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine aswhite foam (21.819 g, 86%).

5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine

[0145]2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine(3.1 g, 4.5 mmol) was dissolved in dry CH₂Cl₂ (4.5 mL) andmethylhydrazine (300 mL 4.64 mmol) was added dropwise at −10° C. to 0°C. After 1 h the mixture was filtered, the filtrate was washed with icecold CH₂Cl₂ and the combined organic phase was washed with water, brineand dried over anhydrous Na₂SO_(4.) The solution was concentrated to get2′-O-(aminooxyethyl) thymidine, which was then dissolved in MeOH (67.5mL). To this formaldehyde (20% aqueous solution, w/w, 1.1 eq.) was addedand the resulting mixture was strirred for 1 h. Solvent was removedunder vacuum; residue chromatographed to get5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine as white foam: (1.95 g, 78%).

5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine

[0146]5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine(1.77 g, 3.12 mmol) was dissolved in a solution of 1M pyridiniump-toluenesulfonate (PPTS) in dry MeOH (30.6 mL). Sodium cyanoborohydride(0.39 g, 6.13 mmol) was added to this solution at 10° C. under inertatmosphere. The reaction mixture was stirred for 10 minutes at 10° C.After that the reaction vessel was removed from the ice bath and stirredat room temperature for 2 h, the reaction monitored by TLC (5% MeOH inCH₂Cl₂). Aqueous NaHCO₃ solution (5%, 10 mL) was added and extractedwith ethyl acetate (2×20 mL). Ethyl acetate phase was dried overanhydrous Na₂SO₄, evaporated to dryness. Residue was dissolved in asolution of 1M PPTS in MeOH (30.6 mL). Formaldehyde (20% w/w, 30 mL,3.37 mmol) was added and the reaction mixture was stirred at roomtemperature for 10 minutes. Reaction mixture cooled to 10° C. in an icebath, sodium cyanoborohydride (0.39 g, 6.13 mmol) was added and reactionmixture stirred at 10° C. for 10 minutes. After 10 minutes, the reactionmixture was removed from the ice bath and stirred at room temperaturefor 2 hrs. To the reaction mixture 5% NaHCO₃ (25 mL) solution was addedand extracted with ethyl acetate (2×25 mL). Ethyl acetate layer wasdried over anhydrous Na₂SO₄ and evaporated to dryness . The residueobtained was purified by flash column chromatography and eluted with 5%MeOH in CH₂Cl₂ to get5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridineas a white foam (14.6 g, 80%).

2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0147] Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was dissolvedin dry THF and triethylamine (1.67 mL, 12 mmol, dry, kept over KOH).This mixture of triethylamine-2HF was then added to5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyll-]5-methyluridine(1.40 g, 2.4 mmol) and stirred at room temperature for 24 hrs. Reactionwas monitored by TLC (5% MeOH in CH₂Cl₂). Solvent was removed undervacuum and the residue placed on a flash column and eluted with 10% MeOHin CH₂Cl₂ to get 2′-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg,92.5%).

5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0148] 2′-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol)was dried over P₂O₅ under high vacuum overnight at 40° C. It was thenco-evaporated with anhydrous pyridine (20 mL). The residue obtained wasdissolved in pyridine (11 mL) under argon atmosphere.4-dimethylaminopyridine (26.5 mg, 2.60 mmol), 4,4′-dimethoxytritylchloride (880 mg, 2.60 mmol) was added to the mixture and the reactionmixture was stirred at room temperature until all of the startingmaterial disappeared. Pyridine was removed under vacuum and the residuechromatographed and eluted with 10% MeOH in CH₂Cl₂ (containing a fewdrops of pyridine) to get5′-O-DMT-2′-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g, 80%).

5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0149] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g,1.67 mmol) was co-evaporated with toluene (20 mL). To the residueN,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) was added and driedover P₂O₅ under high vacuum overnight at 40° C. Then the reactionmixture was dissolved in anhydrous acetonitrile (8.4 mL) and2-cyanoethyl-N,N,N¹,N¹-tetraisopropylphosphoramidite (2.12 mL, 6.08mmol) was added. The reaction mixture was stirred at ambient temperaturefor 4 hrs under inert atmosphere. The progress of the reaction wasmonitored by TLC (hexane:ethyl acetate 1:1). The solvent was evaporated,then the residue was dissolved in ethyl acetate (70 mL) and washed with5% aqueous NaHCO₃ (40 mL). Ethyl acetate layer was dried over anhydrousNa₂SO₄ and concentrated. Residue obtained was chromatographed (ethylacetate as eluent) to get5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]as a foam (1.04 g, 74.9%).

2′-(Aminooxyethoxy) nucleoside amidites

[0150] 2′-(Aminooxyethoxy) nucleoside amidites (also known in the art as2′-O-(aminooxyethyl) nucleoside amidites) are prepared as described inthe following paragraphs. Adenosine, cytidine and thymidine nucleosideamidites are prepared similarly.

N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0151] The 2′-O-aminooxyethyl guanosine analog may be obtained byselective 2′-O-alkylation of diaminopurine riboside. Multigramquantities of diaminopurine riboside may be purchased from Schering AG(Berlin) to provide 2′-O-(2-ethylacetyl) diaminopurine riboside alongwith a minor amount of the 3′-O-isomer. 2′-O-(2-ethylacetyl)diaminopurine riboside may be resolved and converted to2′-O-(2-ethylacetyl)guanosine by treatment with adenosine deaminase.(McGee, D. P. C., Cook, P. D., Guinosso, C. J., WO 94/02501 A1 940203.)Standard protection procedures should afford2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine and2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosinewhich may be reduced to provide2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-hydroxyethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine.As before the hydroxyl group may be displaced by N-hydroxyphthalimidevia a Mitsunobu reaction, and the protected nucleoside mayphosphitylated as usual to yield2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-([2-phthalmidoxy]ethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].

2′-dimethylaminoethoxyethoxy (2′-DMAEOE) nucleoside amidites

[0152] 2′-dimethylaminoethoxyethoxy nucleoside amidites (also known inthe art as 2′-O-dimethylaminoethoxyethyl, i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂,or 2′-DMAEOE nucleoside amidites) are prepared as follows. Othernucleoside amidites are prepared similarly.

2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine

[0153] 2[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol) isslowly added to a solution of borane in tetra-hydrofuran (1 M, 10 mL, 10mmol) with stirring in a 100 mL bomb. Hydrogen gas evolves as the soliddissolves. O²-,2′-anhydro-5-methyluridine (1.2 g, 5 mmol), and sodiumbicarbonate (2.5 mg) are added and the bomb is sealed, placed in an oilbath and heated to 155° C. for 26 hours. The bomb is cooled to roomtemperature and opened. The crude solution is concentrated and theresidue partitioned between water (200 mL) and hexanes (200 mL). Theexcess phenol is extracted into the hexane layer. The aqueous layer isextracted with ethyl acetate (3×200 mL) and the combined organic layersare washed once with water, dried over anhydrous sodium sulfate andconcentrated. The residue is columned on silica gel usingmethanol/methylene chloride 1:20 (which has 2% triethylamine) as theeluent. As the column fractions are concentrated a colorless solid formswhich is collected to give the title compound as a white solid.

5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine

[0154] To 0.5 g (1.3 mmol) of2′-O-[2(2-N,N-dimethylamino-ethoxy)ethyl)]-5-methyl uridine in anhydrouspyridine (8 mL), triethylamine (0.36 mL) and dimethoxytrityl chloride(DMT-Cl, 0.87 g, 2 eq.) are added and stirred for 1 hour. The reactionmixture is poured into water (200 mL) and extracted with CH₂Cl₂ (2×200mL). The combined CH₂Cl₂ layers are washed with saturated NaHCO₃solution, followed by saturated NaCl solution and dried over anhydroussodium sulfate. Evaporation of the solvent followed by silica gelchromatography using MeOH:CH₂Cl₂:Et₃N (20:1, v/v, with 1% triethylamine)gives the title compound.

5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite

[0155] Diisopropylaminotetrazolide (0.6 g) and2-cyanoethoxy-N,N-diisopropyl phosphoramidite (1.1 mL, 2 eq.) are addedto a solution of5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine(2.17 g, 3 mmol) dissolved in CH₂Cl₂ (20 mL) under an atmosphere ofargon. The reaction mixture is stirred overnight and the solventevaporated. The resulting residue is purified by silica gel flash columnchromatography with ethyl acetate as the eluent to give the titlecompound.

Example 2 Oligonucleotide synthesis

[0156] Unsubstituted and substituted phosphodiester (P═O)oligonucleotides are synthesized on an automated DNA synthesizer(Applied Biosystems model 380B) using standard phosphoramidite chemistrywith oxidation by iodine.

[0157] Phosphorothioates (P═S) are synthesized as for the phosphodiesteroligonucleotides except the standard oxidation bottle was replaced by0.2 M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrilefor the stepwise thiation of the phosphite linkages. The thiation waitstep was increased to 68 sec and was followed by the capping step. Aftercleavage from the CPG column and deblocking in concentrated ammoniumhydroxide at 55° C. (18 h), the oligonucleotides were purified byprecipitating twice with 2.5 volumes of ethanol from a 0.5 M NaClsolution. Phosphinate oligonucleotides are prepared as described in U.S.Pat. No. 5,508,270, herein incorporated by reference.

[0158] Alkyl phosphonate oligonucleotides are prepared as described inU.S. Pat. No. 4,469,863, herein incorporated by reference.

[0159] 3′-Deoxy-3′-methylene phosphonate oligonucleotides are preparedas described in U.S. Pat. Nos. 5,610,289 or 5,625,050, hereinincorporated by reference.

[0160] Phosphoramidite oligonucleotides are prepared as described inU.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporatedby reference.

[0161] Alkylphosphonothioate oligonucleotides are prepared as describedin published PCT applications PCT/US94/00902 and PCT/US93/06976(published as WO 94/17093 and WO 94/02499, respectively), hereinincorporated by reference.

[0162] 3′-Deoxy-3′-amino phosphoramidate oligonucleotides are preparedas described in U.S. Pat. No. 5,476,925, herein incorporated byreference.

[0163] Phosphotriester oligonucleotides are prepared as described inU.S. Pat. No. 5,023,243, herein incorporated by reference.

[0164] Borano phosphate oligonucleotides are prepared as described inU.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated byreference.

Example 3 Oligonucleoside synthesis

[0165] Methylenemethylimino linked oligonucleosides, also identified asMMI linked oligonucleosides, methylenedi-methylhydrazo linkedoligonucleosides, also identified as MDH linked oligonucleosides, andmethylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligo-nucleosides, also identified as amide-4 linked oligonucleo-sides,as well as mixed backbone compounds having, for instance, alternatingMMI and P═O or P═S linkages are prepared as described in U.S. Pat. Nos.5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of whichare herein incorporated by reference.

[0166] Formacetal and thioformacetal linked oligonucleosides areprepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, hereinincorporated by reference.

[0167] Ethylene oxide linked oligonucleosides are prepared as describedin U.S. Pat. No. 5,223,618, herein incorporated by reference.

Example 4 PNA Synthesis

[0168] Peptide nucleic acids (PNAs) are prepared in accordance with anyof the various procedures referred to in Peptide Nucleic Acids (PNA):Synthesis, Properties and Potential Applications, Bioorganic & MedicinalChemistry, 1996, 4, 5-23. They may also be prepared in accordance withU.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262, herein incorporatedby reference.

Example 5 Synthesis of Chimeric Oligonucleotides

[0169] Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the “gap” segment oflinked nucleosides is positioned between 5′ and 3′ “wing” segments oflinked nucleosides and a second “open end” type wherein the “gap”segment is located at either the 3′ or the 5′ terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas “gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers”.

[2′-O-Me]—[2′-deoxy—[2′-O-Me]Chimeric Phosphorothioate Oligonucleotides

[0170] Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and2′-deoxy phosphorothioate oligo-nucleotide segments are synthesizedusing an Applied Biosystems automated DNA synthesizer Model 380B, asabove. Oligonucleotides are synthesized using the automated synthesizerand 2′-deoxy-5′-dimethoxytrityl-3-O-phosphor-amidite for the DNA portionand 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′wings. The standard synthesis cycle is modified by increasing the waitstep after the delivery of tetrazole and base to 600 s repeated fourtimes for RNA and twice for 2′-O-methyl. The fully protectedoligonucleotide is cleaved from the support and the phosphate group isdeprotected in 3:1 ammonia/ethanol at room temperature overnight thenlyophilized to dryness. Treatment in methanolic ammonia for 24 hrs atroom temperature is then done to deprotect all bases and sample wasagain lyophilized to dryness. The pellet is resuspended in 1M TBAF inTHF for 24 hrs at room temperature to deprotect the 2′ positions. Thereaction is then quenched with 1M TEAA and the sample is then reduced to½ volume by rotovac before being desalted on a G25 size exclusioncolumn. The oligo recovered is then analyzed spectrophotometrically foryield and for purity by capillary electrophoresis and by massspectrometry.

[2′-O-(2-Methoxyethyl)]—[2′-deoxy]—[2′-O-(Methoxyethyl)]ChimericPhosphorothioate. Oligonucleotides

[0171][2′-O-(2-methoxyethyl)]—[2′-deoxy]—[-2′-O-(methoxy-ethyl)]chimericphosphorothioate oligonucleotides were prepared as per the procedureabove for the 2′-O-methyl chimeric oligonucleotide, with thesubstitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methylamidites.

[2′-O-(2-Methoxyethyl)Phosphodiester]—[2′-deoxyPhosphorothioate]—[2′-O-(2-Methoxyethyl). Phosphodiester]ChimericOligonucleotides

[0172] [2′-O-(2-methoxyethyl phosphodiester]—[2′-deoxyphos-phorothioate]—[2′-O-(methoxyethyl)phosphodiester]chimericoligonucleotides are prepared as per the above procedure for the2′-O-methyl chimeric oligonucleotide with the substitution of2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidizationwith iodine to generate the phosphodiester internucleotide linkageswithin the wing portions of the chimeric structures and sulfurizationutilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) togenerate the phosphorothioate internucleotide linkages for the centergap.

[0173] Other chimeric oligonucleotides, chimeric oligonucleo-sides andmixed chimeric oligonucleotides/oligonucleosides are synthesizedaccording to U.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 6 Oligonucleotide Isolation

[0174] After cleavage from the controlled pore glass column (AppliedBiosystems) and deblocking in concentrated ammonium hydroxide at 55° C.for 18 hours, the oligonucleotides or oligonucleosides are purified byprecipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol.Synthesized oligonucleotides were analyzed by polyacrylamide gelelectrophoresis on denaturing gels and judged to be at least 85% fulllength material. The relative amounts of phosphorothioate andphosphodiester linkages obtained in synthesis were periodically checkedby ³¹p nuclear magnetic resonance spectroscopy, and for some studiesoligonucleotides were purified by HPLC, as described by Chiang et al.,J. Biol. Chem. 1991, 266, 18162-18171. Results obtained withHPLC-purified material were similar to those obtained with non-HPLCpurified material.

Example 7 Oligonucleotide Synthesis—96 Well Plate Format

[0175] Oligonucleotides were synthesized via solid phase P(III)phosphoramidite chemistry on an automated synthesizer capable ofassembling 96 sequences simultaneously in a standard 96 well format.Phosphodiester internucleotide linkages were afforded by oxidation withaqueous iodine. Phosphorothioate internucleotide linkages were generatedby sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyldiisopropyl phosphoramidites were purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per known literature or patented methods. They are utilized as baseprotected beta-cyanoethyldiisopropyl phosphoramidites.

[0176] Oligonucleotides were cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product was thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 8 Oligonucleotide Analysis—96 Well Plate Format

[0177] The concentration of oligonucleotide in each well was assessed bydilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products was evaluated by capillaryelectrophoresis (CE) in either the 96 well format (Beckman P/ACE™ MDQ)or, for individually prepared samples, on a commercial CE apparatus(e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition wasconfirmed by mass analysis of the compounds utilizing electrospray-massspectroscopy. All assay test plates were diluted from the master plateusing single and multi-channel robotic pipettors. Plates were judged tobe acceptable if at least 85% of the compounds on the plate were atleast 85% full length.

Example 9 Cell Culture and Oligonucleotide Treatment

[0178] The effect of antisense compounds on target nucleic acidexpression can be tested in any of a variety of cell types provided thatthe target nucleic acid is present at measurable levels. This can beroutinely determined using, for example, PCR or Northern blot analysis.The following 7 cell types are provided for illustrative purposes, butother cell types can be routinely used, provided that the target isexpressed in the cell type chosen. This can be readily determined bymethods routine in the art, for example Northern blot analysis,Ribonuclease protection assays, or RT-PCR.

[0179] T-24 Cells:

[0180] The human transitional cell bladder carcinoma cell line T-24 wasobtained from the American Type Culture Collection (ATCC) (Manassas,Va.). T-24 cells were routinely cultured in complete McCoy's 5A basalmedia (Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10%fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.),penicillin 100 units per mL, and streptomycin 100 micrograms per mL(Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinelypassaged by trypsinization and dilution when they reached 90%confluence. Cells were seeded into 96-well plates (Falcon-Primaria#3872) at a density of 7000 cells/well for use in RT-PCR analysis.

[0181] For Northern blotting or other analysis, cells may be seeded onto100 mm or other standard tissue culture plates and treated similarly,using appropriate volumes of medium and oligonucleotide.

[0182] A549 cells:

[0183] The human lung carcinoma cell line A549 was obtained from theAmerican Type Culture Collection (ATCC) (Manassas, Va.). A549 cells wereroutinely cultured in DMEM basal media (Gibco/Life Technologies,Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/LifeTechnologies, Gaithersburg, Md.), penicillin 100 units per mL, andstreptomycin 100 micrograms per mL (Gibco/Life Technologies,Gaithersburg, Md.). Cells were routinely passaged by trypsinization anddilution when they reached 90% confluence.

[0184] NHDF Cells:

[0185] Human neonatal dermal fibroblast (NHDF) were obtained from theClonetics Corporation (Walkersville Md.). NHDFs were routinelymaintained in Fibroblast Growth Medium (Clonetics Corporation,Walkersville Md.) supplemented as recommended by the supplier. Cellswere maintained for up to 10 passages as recommended by the supplier.

[0186] HEK Cells:

[0187] Human embryonic keratinocytes (HEK) were obtained from theClonetics Corporation (Walkersville, Md.). HEKs were routinelymaintained in Keratinocyte Growth Medium (Clonetics Corporation,Walkersville Md.) formulated as recommended by the supplier. Cells wereroutinely maintained for up to 10 passages as recommended by thesupplier.

[0188] HepG2 Cells:

[0189] The human hepatoblastoma cell line HepG2 was obtained from theAmerican Type Culture Collection (Manassas, Va.). HepG2 cells wereroutinely cultured in Eagle′s MEM supplemented with 10% fetal calfserum, non-essential amino acids, and 1 mM sodium pyruvate (Gibco/LifeTechnologies, Gaithersburg, Md.). Cells were routinely passaged bytrypsinization and dilution when they reached 90% confluence. Cells wereseeded into 96-well plates (Falcon-Primaria #3872) at a density of 7000cells/well for use in RT-PCR analysis.

[0190] For Northern blotting or other analyses, cells may be seeded onto100 mm or other standard tissue culture plates and treated similarly,using appropriate volumes of medium and oligonucleotide.

[0191] AML12 Cells:

[0192] AML12 (alpha mouse liver 12) cell line was established fromhepatocytes from a mouse (CD1 strain, line MT42) transgenic for humanTGF alpha. Cells are cultured in a 1:1 mixture of Dulbecco's modifiedEagle's medium and Ham's F12 medium with 0.005 mg/ml insulin, 0.005mg/ml transferrin, 5 ng/ml selenium, and 40 ng/ml dexamethasone, and90%; 10% fetal bovine serum. For subculturing, spent medium is removedand fresh media of 0.25% trypsin, 0.03% EDTA solution is added. Freshtrypsin solution (1 to 2 ml) is added and the culture is left to sit atroom temperature until the cells detach.

[0193] Cells were routinely passaged by trypsinization and dilution whenthey reached 90% confluence. Cells were seeded into 96-well plates(Falcon-Primaria #3872) at a density of 7000 cells/well for use inRT-PCR analysis.

[0194] For Northern blotting or other analyses, cells may be seeded onto100 mm or other standard tissue culture plates and treated similarly,using appropriate volumes of medium and oligonucleotide.

Primary Mouse Hepatocytes:

[0195] Primary mouse hepatocytes were prepared from CD-1 mice purchasedfrom Charles River Labs (Wilmington, Mass.) and were routinely culturedin Hepatoyte Attachment Media (Gibco) supplemented with 10Fetal BovineSerum (Gibco/Life Technologies, Gaithersburg, Md.), 250 nM dexamethasone(Sigma), and 10 nM bovine insulin (Sigma). Cells were seeded into96-well plates (Falcon-Primaria #3872) at a density of 10000 cells/wellfor use in RT-PCR analysis.

[0196] For Northern blotting or other analyses, cells are plated onto100 mm or other standard tissue culture plates coated with rat tailcollagen (200 ug/mL) (Becton Dickinson) and treated similarly usingappropriate volumes of medium and oligonucleotide.

[0197] Treatment with Antisense Compounds:

[0198] When cells reach 80% confluency, they are treated witholigonucleotide. For cells grown in 96-well plates, wells are washedonce with 200 μL OPTI-MEM™-1 reduced-serum medium (Gibco BRL) and thentreated with 130 μL of OPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™(Gibco BRL) and the desired concentration of oligonucleotide. After 4-7hours of treatment, the medium is replaced with fresh medium. Cells areharvested 16-24 hours after oligonucleotide treatment.

[0199] The concentration of oligonucleotide used varies from cell lineto cell line. To determine the optimal oligonucleotide concentration fora particular cell line, the: cells are treated with a positive controloligonucleotide at a range of concentrations. For human cells thepositive control oligonucleotide is ISIS 13920, TCCGTCATCGCTCCTCAGGG,SEQ ID NO: 1, a 2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown inbold) with a phosphorothioate backbone which is targeted to human H-ras.For mouse or rat cells the positive control oligonucleotide is ISIS15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 2, a 2′-O-methoxyethyl gapmer(2′-O-methoxyethyls shown in bold) with a phosphorothioate backbonewhich is targeted to both mouse and rat c-raf. The concentration ofpositive control oligonucleotide that results in 80% inhibition ofc-Ha-ras (for ISIS 13920) or c-raf (for ISIS 15770) mRNA is thenutilized as the screening concentration for new oligonucleotides insubsequent experiments for that cell line. If 80% inhibition is notachieved, the lowest concentration of positive control oligonucleotidethat results in 60% inhibition of H-ras or c-raf mRNA is then utilizedas the oligonucleotide screening concentration in subsequent experimentsfor that cell line. If 60% inhibition is not achieved, that particularcell line is deemed as unsuitable for oligonucleotide transfectionexperiments.

Example 10 Analysis of oligonucleotide Inhibition of apolipoprotein(a)Expression

[0200] Antisense modulation of apolipoprotein(a) expression can beassayed in a variety of ways known in the art. For example,apolipoprotein(a) mRNA levels can be quantitated by, e.g., Northern blotanalysis, competitive polymerase chain reaction (PCR), or real-time PCR(RT-PCR). Real-time quantitative PCR is presently preferred. RNAanalysis can be performed on total cellular RNA or poly(A)+ mRNA.Methods of RNA isolation are taught in, for example, Ausubel, F. M. etal., Current Protocols in Molecular Biology, Volume 1, pp. 4.1.1-4.2.9and 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993. Northern blot analysisis routine in the art and is taught in, for example, Ausubel, F. M. etal., Current Protocols in Molecular Biology, Volume 1, pp. 4.2.1-4.2.9,John Wiley & Sons, Inc., 1996. Real-time quantitative (PCR) can beconveniently accomplished using the commercially available ABI PRISM™7700 Sequence Detection System, available from PE-Applied Biosystems,Foster City, Calif. and used according to manufacturer's instructions.

[0201] Protein levels of apolipoprotein(a) can be quantitated in avariety of ways well known in the art, such as immunoprecipitation,Western blot analysis (immunoblotting), ELISA or fluorescence-activatedcell sorting (FACS). Antibodies directed to apolipoprotein(a) can beidentified and obtained from a variety of sources, such as the MSRScatalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can beprepared via conventional antibody generation methods. Methods forpreparation of polyclonal-antisera are taught in, for example, Ausubel,F. M. et al., Current Protocols in Molecular Biology, Volume 2, pp.11.12.1-11.12.9, John Wiley & Sons, Inc., 1997. Preparation ofmonoclonal antibodies is taught in, for example, Ausubel, F. M. et al.,Current Protocols in Molecular Biology, Volume 2, pp. 11.4.1-11.11.5,John Wiley & Sons, Inc., 1997.

[0202] Immunoprecipitation methods are standard in the art and can befound at, for example, Ausubel, F. M. et al., Current Protocols inMolecular Biology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons,Inc., 1998. Western blot (immunoblot) analysis is standard in the artand can be found at, for example, Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley& Sons, Inc., 1997. Enzyme-linked immunosorbent assays (ELISA) arestandard in the art and can be found at, for example, Ausubel, F. M. etal., Current Protocols in Molecular Biology, Volume 2, pp.11.2.1-11.2.22, John Wiley & Sons, Inc., 1991.

Example 11 Poly(A)+ mRNA isolation

[0203] Poly(A)+ mRNA can be isolated according to Miura et al., Clin.Chem., 1996, 42, 1758-1764. Other methods for poly(A)+ mRNA isolationare taught in, for example, Ausubel, F. M. et al., Current Protocols inMolecular Biology, Volume 1, pp. 4.5.1-4.5.3, John Wiley & Sons, Inc.,1993. Briefly, for cells grown on 96-well plates, growth medium isremoved from the cells and each well is washed with 200 μL cold PBS. 60μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5%NP-40, 20 mM vanadyl-ribonucleoside complex) is added to each well, theplate is gently agitated and then incubated at room temperature for fiveminutes. 55 μL of lysate is transferred to Oligo d(T) coated 96-wellplates (AGCT Inc., Irvine Calif.). Plates are incubated for 60 minutesat room temperature, washed 3 times with 200 μL of wash buffer (10 mMTris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the plateis blotted on paper towels to remove excess wash buffer and thenair-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6),preheated to 70° C. is added to each well, the plate is incubated on a90° C. hot plate for 5 minutes, and the eluate is then transferred to afresh 96-well plate.

[0204] Cells grown on 100 mm or other standard plates may be treatedsimilarly, using appropriate volumes of all solutions.

Example 12 Total RNA Isolation

[0205] Total RNA can be isolated using an RNEASY 96™ kit and bufferspurchased from Qiagen Inc. (Valencia Calif.) following themanufacturer's recommended procedures. Briefly, for cells grown on96-well plates, growth medium is removed from the cells and each well iswashed with 200 μL cold PBS. 100 μL Buffer RLT is added to each well andthe plate vigorously agitated for 20 seconds. 100 μL of 70% ethanol isthen added to each well and the contents mixed by pipetting three timesup and down. The samples are then transferred to the RNEASY 96™ wellplate attached to a QIAVAC™ manifold fitted with a waste collection trayand attached to a vacuum source. Vacuum is applied for 15 seconds. 1 mLof Buffer RW1 was added to each well of the RNEASY 96™ plate and thevacuum again applied for 15 seconds. 1 mL of Buffer RPE is then added toeach well of the RNEASY 96™ plate and the vacuum applied for a period of15 seconds. The Buffer RPE wash was then repeated and the vacuum isapplied for an additional 10 minutes. The plate is then removed from theQIAVAC™ manifold and blotted dry on paper towels. The plate is thenre-attached to the QIAVAC™ manifold fitted with a collection tube rackcontaining 1.2 mL collection tubes. RNA is then eluted by pipetting 60μL water into each well, incubating 1 minute, and then applying thevacuum for 30 seconds. The elution step is repeated with an additional60 μL water.

[0206] The repetitive pipetting and elution steps may be automated usinga QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially,after lysing of the cells on the culture plate, the plate is transferredto the robot deck where the pipetting, DNase treatment and elution stepsare carried out.

Example 13 Real-Time Quantitative PCR Analysis of apolipoprotein(a) mRNALevels

[0207] Quantitation of apolipoprotein(a) mRNA levels can be determinedby real-time quantitative PCR using the ABI PRISM™ 7700 SequenceDetection System (PE-Applied Biosystems, Foster City, Calif.) accordingto manufacturer's instructions. This is a closed-tube, non-gel-based,fluorescence detection system which allows high-throughput quantitationof polymerase chain reaction (PCR) products in real-time. As opposed tostandard PCR, in which amplification products are quantitated after thePCR is completed, products in real-time quantitative PCR are quantitatedas they accumulate. This is accomplished by including in the PCRreaction an oligonucleotide probe that anneals specifically between theforward and reverse PCR primers, and contains two fluorescent dyes. Areporter dye (e.g., JOE, FAM, or VIC, obtained from either OperonTechnologies Inc., Alameda, Calif. or PE-Applied Biosystems, FosterCity, Calif.) is attached to the 5′ end of the probe and a quencher dye(e.g., TAMRA, obtained from either Operon Technologies Inc., Alameda,Calif. or PE-Applied Biosystems, Foster City, Calif.) is attached to the3′ end of the probe. When the probe and dyes are intact, reporter dyeemission is quenched by the proximity of the 3′ quencher dye. Duringamplification, annealing of the probe to the target sequence creates asubstrate that can be cleaved by the 5′-exonuclease activity of Taqpolymerase. During the extension phase of the PCR amplification cycle,cleavage of the probe by Taq polymerase releases the reporter dye fromthe remainder of the probe (and hence from the quencher moiety) and asequence-specific fluorescent signal is generated. With each cycle,additional reporter dye molecules are cleaved from their respectiveprobes, and the fluorescence intensity is monitored at regular intervalsby laser optics built into the ABI PRISMS ™ 7700 Sequence DetectionSystem. In each assay, a series of parallel reactions containing serialdilutions of mRNA from untreated control samples generates a standardcurve that is used to quantitate the percent inhibition after antisenseoligonucleotide treatment of test samples.

[0208] Prior to quantitative PCR analysis, primer-probe sets specific tothe target gene being measured are evaluated for their ability to be“multiplexed” with a GAPDH amplification reaction. In multiplexing, boththe target gene and the internal standard gene GAPDH are amplifiedconcurrently in a single sample. In this analysis, mRNA isolated fromuntreated cells is serially diluted. Each dilution is amplified in thepresence of primer-probe sets specific for GAPDH only, target gene only(“single-plexing”), or both (multiplexing). Following PCR amplification,standard curves of GAPDH and target mRNA signal as a function ofdilution are generated from both the single-plexed and multiplexedsamples. If both the slope and correlation coefficient of the GAPDH andtarget signals generated from the multiplexed samples fall within 10% oftheir corresponding values generated from the single-plexed samples, theprimer-probe set specific for that target is deemed multiplexable. Othermethods of PCR are also known in the art.

[0209] PCR reagents are obtained from PE-Applied Biosystems, FosterCity, Calif. RT-PCR reactions are carried out by adding 25 μL PCRcocktail (1×TAQMAN™ buffer A, 5.5 mM MgCl₂, 300 μM each of dATP, dCTPand dGTP, 600 μM of dUTP, 100 nM each of forward primer, reverse primer,and probe, 20 Units RNAse inhibitor, 1.25 Units AMPLITAQ GOLD™, and 12.5Units MuLV reverse transcriptase) to 96 well plates containing 25 μLtotal RNA solution. The RT reaction is carried out by incubation for 30minutes at 48° C. Following a 10 minute incubation at 95° C. to activatethe AMPLITAQ GOLD™, 40 cycles of a two-step PCR protocol are carriedout: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5minutes (annealing/extension).

[0210] Gene target quantities obtained by real time RT-PCR can benormalized using either the expression level of GAPDH, a gene whoseexpression is constant, or by quantifying total RNA using RiboGreen™(Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantifiedby real time RT-PCR, by being run simultaneously with the target,multiplexing, or separately. Total RNA is quantified using RiboGreen™RNA quantification reagent from Molecular Probes (Eugene, Oreg.).Methods of RNA quantification by RiboGreen™ are taught in Jones, L. J.,et al, Analytical Biochemistry, 1998, 265, 368-374.

[0211] In this assay, 175 μL of RiboGreen™ working reagent (RiboGreen™reagent diluted 1:2865 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipettedinto a 96-well plate containing 25 uL purified, cellular RNA. The plateis read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at480 nm and emission at 520 nm.

[0212] Probes and primers to human apolipoprotein(a) are designed tohybridize to a human apolipoprotein(a) sequence, using publishedsequence information (GenBank accession number NM_(—)005577,incorporated herein as SEQ ID NO: 3).

[0213] For human GAPDH the standard PCR primers are: forward primer:GAAGGTGAAGGTCGGAGTC (SEQ ID NO: 4) reverse primer: GAAGATGGTGATGGGATTTC(SEQ ID NO: 5) and the PCR probe is: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA3′ (SEQ ID NO: 6) where JOE (PE-Applied Biosystems, Foster City, Calif.)is the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems,Foster City, Calif.) is the quencher dye.

Example 14 Northern Blot Analysis of apolipoprotein(a) mRNA Levels

[0214] Eighteen hours after antisense treatment, cell monolayers arewashed twice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc.,Friendswood, Tex.). Total RNA is prepared following manufacturer'srecommended protocols. Twenty micrograms of total RNA is fractionated byelectrophoresis through 1.2% agarose gels containing 1.1% formaldehydeusing a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNA istransferred from the gel to HYBOND™-N+ nylon membranes (AmershamPharmacia Biotech, Piscataway, N.J.) by overnight capillary transferusing a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc.,Friendswood, Tex.). RNA transfer is confirmed by UV visualization.Membranes are fixed by UV cross-linking using a STRATALINKER™ UVCrosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probedusing QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.)using manufacturer's recommendations for stringent conditions.

[0215] To detect human apolipoprotein(a), a human apolipoprotein(a)specific probe is prepared by PCR using a forward and a reverse primer.To normalize for variations in loading and transfer efficiency,membranes are stripped and probed for human glyceraldehyde-3-phosphatedehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).

[0216] Hybridized membranes are visualized and quantitated using aPHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics,Sunnyvale, Calif.). Data are normalized to GAPDH levels in untreatedcontrols.

Example 15 Chimeric phosphorothioate oligonucleotides having 2′-MOEwings and a deoxy gap targeting human apolipoprotein(a)

[0217] In accordance with the present invention, a series ofoligonucleotides were designed to target different regions of the humanapolipoprotein(a) RNA, using published sequence (GenBank accessionnumber NM_(—)005577, incorporated herein as SEQ ID NO: 3). Theoligonucleotides are shown in Table 1. “Target site” indicates the first(5′-most) nucleotide number on the particular target sequence to whichthe oligonucleotide binds. All compounds in Table 1 are chimericoligonucleotides (“gapmers”) 20 nucleotides in length, composed of acentral “gap” region consisting of ten 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′directions) by five-nucleotide “wings”.The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines. TABLE 1 Chimeric phosphorothioate oligonucleotideshaving 2′-MOE wings and a deoxy gap targeting human apolipoprotein(a)TARGET SEQ SEQ ID TARGET ID ISIS # REGION NO SITE SEQUENCE NO 144367Coding 3 174 GGCAGGTCCTTCCTGTGACA 7 144368 Coding 3 352TCTGCGTCTGAGCATTGCGT 8 144369 Coding 3 522 AAGCTTGGCAGGTTCTTCCT 9 144370Coding 3 1743 TCGGAGGCGCGACGGCAGTC 10 144371 Coding 3 2768CGGAGGCGCGACGGCAGTCC 11 144372 Coding 3 2910 GGCAGGTTCTTCCTGTGACA 12144373 Coding 3 3371 ATAACAATAAGGAGCTGCCA 13 144374 Coding 3 4972GACCAAGCTTGGCAGGTTCT 14 144375 Coding 3 5080 TAACAATAAGGAGCTGCCAC 15144376 Coding 3 5315 TGACCAAGCTTGGCAGGTTC 16 144377 Coding 3 5825TTCTGCGTCTGAGCATTGCG 17 144378 Coding 3 6447 AACAATAAGGAGCTGCCACA 18144379 Coding 3 7155 ACCTGACACCGGGATCCCTC 19 144380 Coding 3 7185CTGAGCATTGCGTCAGGTTG 20 144381 Coding 3 8463 AGTAGTTCATGATCAAGCCA 21144382 Coding 3 8915 GACGGCAGTCCCTTCTGCGT 22 144383 Coding 3 9066GGCAGGTTCTTCCAGTGACA 23 144384 Coding 3 10787 TGACCAAGCTTGGCAAGTTC 24144385 Coding 3 11238 TATAACACCAAGGACTAATC 25 144386 Coding 3 11261CCATCTGACATTGGGATCCA 26 144387 Coding 3 11461 TGTGGTGTCATAGAGGACCA 27144388 Coding 3 11823 ATGGGATCCTCCGATGCCAA 28 144389 Coding 3 11894ACACCAAGGGCGAATCTCAG 29 144390 Coding 3 11957 TTCTGTCACTGGACATCGTG 30144391 Coding 3 12255 CACACGGATCGGTTGTGTAA 31 144392 Coding 3 12461ACATGTCCTTCCTGTGACAG 32 144393 Coding 3 12699 CAGAAGGAGGCCCTAGGCTT 33144394 Coding 3 13354 CTGGCGGTGACCATGTAGTC 34 144395 3′UTR 3 13711TCTAAGTAGGTTGATGCTTC 35 144396 3′UTR 3 13731 TCCTTACCCACGTTTCAGCT 36144397 3′UTR 3 13780 GGAACAGTGTCTTCGTTTGA 37 144398 3′UTR 3 13801GTTTGGCATAGCTGGTAGCT 38 144399 3′UTR 3 13841 ACCTTAAAAGCTTATACACA 39144400 3′UTR 3 13861 ATACAGAATTTGTCAGTCAG 40 144401 3′UTR 3 13881GTCATAGCTATGACACCTTA 41

Example 16 Western Blot Analysis of apolipoprotein(a) Protein Levels

[0218] Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16-20 h after oligonucleotidetreatment, washed once with PBS, suspended in Laemmli buffer (100ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gelsare run for 1.5 hours at 150 V, and transferred to membrane for westernblotting. Appropriate primary antibody directed to apolipoprotein(a) isused, with a radiolabelled or fluorescently labeled secondary antibodydirected against the primary antibody species. Bands arevisualized,using a PHOSPHORIMAGER™ (Molecular Dynamics, SunnyvaleCalif.).

1 41 1 20 DNA Artificial Sequence Antisense Oligonucleotide 1 tccgtcatcgctcctcaggg 20 2 20 DNA Artificial Sequence Antisense Oligonucleotide 2atgcattctg cccccaagga 20 3 13938 DNA Homo sapiens CDS (46)...(13692) 3ctgggattgg gacacacttt ctggacactg ctggccagtc ccaaa atg gaa cat aag 57 MetGlu His Lys 1 gaa gtg gtt ctt cta ctt ctt tta ttt ctg aaa tca gca gcacct gag 105 Glu Val Val Leu Leu Leu Leu Leu Phe Leu Lys Ser Ala Ala ProGlu 5 10 15 20 caa agc cat gtg gtc cag gat tgc tac cat ggt gat gga cagagt tat 153 Gln Ser His Val Val Gln Asp Cys Tyr His Gly Asp Gly Gln SerTyr 25 30 35 cga ggc acg tac tcc acc act gtc aca gga agg acc tgc caa gcttgg 201 Arg Gly Thr Tyr Ser Thr Thr Val Thr Gly Arg Thr Cys Gln Ala Trp40 45 50 tca tct atg aca cca cat caa cat aat agg acc aca gaa aac tac cca249 Ser Ser Met Thr Pro His Gln His Asn Arg Thr Thr Glu Asn Tyr Pro 5560 65 aat gct ggc ttg atc atg aac tac tgc agg aat cca gat gct gtg gca297 Asn Ala Gly Leu Ile Met Asn Tyr Cys Arg Asn Pro Asp Ala Val Ala 7075 80 gct cct tat tgt tat acg agg gat ccc ggt gtc agg tgg gag tac tgc345 Ala Pro Tyr Cys Tyr Thr Arg Asp Pro Gly Val Arg Trp Glu Tyr Cys 8590 95 100 aac ctg acg caa tgc tca gac gca gaa ggg act gcc gtc gcg cctccg 393 Asn Leu Thr Gln Cys Ser Asp Ala Glu Gly Thr Ala Val Ala Pro Pro105 110 115 act gtt acc ccg gtt cca agc cta gag gct cct tcc gaa caa gcaccg 441 Thr Val Thr Pro Val Pro Ser Leu Glu Ala Pro Ser Glu Gln Ala Pro120 125 130 act gag caa agg cct ggg gtg cag gag tgc tac cat ggt aat ggacag 489 Thr Glu Gln Arg Pro Gly Val Gln Glu Cys Tyr His Gly Asn Gly Gln135 140 145 agt tat cga ggc aca tac tcc acc act gtc aca gga aga acc tgccaa 537 Ser Tyr Arg Gly Thr Tyr Ser Thr Thr Val Thr Gly Arg Thr Cys Gln150 155 160 gct tgg tca tct atg aca cca cac tcg cat agt cgg acc cca gaatac 585 Ala Trp Ser Ser Met Thr Pro His Ser His Ser Arg Thr Pro Glu Tyr165 170 175 180 tac cca aat gct ggc ttg atc atg aac tac tgc agg aat ccagat gct 633 Tyr Pro Asn Ala Gly Leu Ile Met Asn Tyr Cys Arg Asn Pro AspAla 185 190 195 gtg gca gct cct tat tgt tat acg agg gat ccc ggt gtc aggtgg gag 681 Val Ala Ala Pro Tyr Cys Tyr Thr Arg Asp Pro Gly Val Arg TrpGlu 200 205 210 tac tgc aac ctg acg caa tgc tca gac gca gaa ggg act gccgtc gcg 729 Tyr Cys Asn Leu Thr Gln Cys Ser Asp Ala Glu Gly Thr Ala ValAla 215 220 225 cct ccg act gtt acc ccg gtt cca agc cta gag gct cct tccgaa caa 777 Pro Pro Thr Val Thr Pro Val Pro Ser Leu Glu Ala Pro Ser GluGln 230 235 240 gca ccg act gag caa agg cct ggg gtg cag gag tgc tac catggt aat 825 Ala Pro Thr Glu Gln Arg Pro Gly Val Gln Glu Cys Tyr His GlyAsn 245 250 255 260 gga cag agt tat cga ggc aca tac tcc acc act gtc acagga aga acc 873 Gly Gln Ser Tyr Arg Gly Thr Tyr Ser Thr Thr Val Thr GlyArg Thr 265 270 275 tgc caa gct tgg tca tct atg aca cca cac tcg cat agtcgg acc cca 921 Cys Gln Ala Trp Ser Ser Met Thr Pro His Ser His Ser ArgThr Pro 280 285 290 gaa tac tac cca aat gct ggc ttg atc atg aac tac tgcagg aat cca 969 Glu Tyr Tyr Pro Asn Ala Gly Leu Ile Met Asn Tyr Cys ArgAsn Pro 295 300 305 gat gct gtg gca gct cct tat tgt tat acg agg gat cccggt gtc agg 1017 Asp Ala Val Ala Ala Pro Tyr Cys Tyr Thr Arg Asp Pro GlyVal Arg 310 315 320 tgg gag tac tgc aac ctg acg caa tgc tca gac gca gaaggg act gcc 1065 Trp Glu Tyr Cys Asn Leu Thr Gln Cys Ser Asp Ala Glu GlyThr Ala 325 330 335 340 gtc gcg cct ccg act gtt acc ccg gtt cca agc ctagag gct cct tcc 1113 Val Ala Pro Pro Thr Val Thr Pro Val Pro Ser Leu GluAla Pro Ser 345 350 355 gaa caa gca ccg act gag caa agg cct ggg gtg caggag tgc tac cat 1161 Glu Gln Ala Pro Thr Glu Gln Arg Pro Gly Val Gln GluCys Tyr His 360 365 370 ggt aat gga cag agt tat cga ggc aca tac tcc accact gtc aca gga 1209 Gly Asn Gly Gln Ser Tyr Arg Gly Thr Tyr Ser Thr ThrVal Thr Gly 375 380 385 aga acc tgc caa gct tgg tca tct atg aca cca cactcg cat agt cgg 1257 Arg Thr Cys Gln Ala Trp Ser Ser Met Thr Pro His SerHis Ser Arg 390 395 400 acc cca gaa tac tac cca aat gct ggc ttg atc atgaac tac tgc agg 1305 Thr Pro Glu Tyr Tyr Pro Asn Ala Gly Leu Ile Met AsnTyr Cys Arg 405 410 415 420 aat cca gat gct gtg gca gct cct tat tgt tatacg agg gat ccc ggt 1353 Asn Pro Asp Ala Val Ala Ala Pro Tyr Cys Tyr ThrArg Asp Pro Gly 425 430 435 gtc agg tgg gag tac tgc aac ctg acg caa tgctca gac gca gaa ggg 1401 Val Arg Trp Glu Tyr Cys Asn Leu Thr Gln Cys SerAsp Ala Glu Gly 440 445 450 act gcc gtc gcg cct ccg act gtt acc ccg gttcca agc cta gag gct 1449 Thr Ala Val Ala Pro Pro Thr Val Thr Pro Val ProSer Leu Glu Ala 455 460 465 cct tcc gaa caa gca ccg act gag caa agg cctggg gtg cag gag tgc 1497 Pro Ser Glu Gln Ala Pro Thr Glu Gln Arg Pro GlyVal Gln Glu Cys 470 475 480 tac cat ggt aat gga cag agt tat cga ggc acatac tcc acc act gtc 1545 Tyr His Gly Asn Gly Gln Ser Tyr Arg Gly Thr TyrSer Thr Thr Val 485 490 495 500 aca gga aga acc tgc caa gct tgg tca tctatg aca cca cac tcg cat 1593 Thr Gly Arg Thr Cys Gln Ala Trp Ser Ser MetThr Pro His Ser His 505 510 515 agt cgg acc cca gaa tac tac cca aat gctggc ttg atc atg aac tac 1641 Ser Arg Thr Pro Glu Tyr Tyr Pro Asn Ala GlyLeu Ile Met Asn Tyr 520 525 530 tgc agg aat cca gat gct gtg gca gct ccttat tgt tat acg agg gat 1689 Cys Arg Asn Pro Asp Ala Val Ala Ala Pro TyrCys Tyr Thr Arg Asp 535 540 545 ccc ggt gtc agg tgg gag tac tgc aac ctgacg caa tgc tca gac gca 1737 Pro Gly Val Arg Trp Glu Tyr Cys Asn Leu ThrGln Cys Ser Asp Ala 550 555 560 gaa ggg act gcc gtc gcg cct ccg act gttacc ccg gtt cca agc cta 1785 Glu Gly Thr Ala Val Ala Pro Pro Thr Val ThrPro Val Pro Ser Leu 565 570 575 580 gag gct cct tcc gaa caa gca ccg actgag caa agg cct ggg gtg cag 1833 Glu Ala Pro Ser Glu Gln Ala Pro Thr GluGln Arg Pro Gly Val Gln 585 590 595 gag tgc tac cat ggt aat gga cag agttat cga ggc aca tac tcc acc 1881 Glu Cys Tyr His Gly Asn Gly Gln Ser TyrArg Gly Thr Tyr Ser Thr 600 605 610 act gtc aca gga aga acc tgc caa gcttgg tca tct atg aca cca cac 1929 Thr Val Thr Gly Arg Thr Cys Gln Ala TrpSer Ser Met Thr Pro His 615 620 625 tcg cat agt cgg acc cca gaa tac taccca aat gct ggc ttg atc atg 1977 Ser His Ser Arg Thr Pro Glu Tyr Tyr ProAsn Ala Gly Leu Ile Met 630 635 640 aac tac tgc agg aat cca gat gct gtggca gct cct tat tgt tat acg 2025 Asn Tyr Cys Arg Asn Pro Asp Ala Val AlaAla Pro Tyr Cys Tyr Thr 645 650 655 660 agg gat ccc ggt gtc agg tgg gagtac tgc aac ctg acg caa tgc tca 2073 Arg Asp Pro Gly Val Arg Trp Glu TyrCys Asn Leu Thr Gln Cys Ser 665 670 675 gac gca gaa ggg act gcc gtc gcgcct ccg act gtt acc ccg gtt cca 2121 Asp Ala Glu Gly Thr Ala Val Ala ProPro Thr Val Thr Pro Val Pro 680 685 690 agc cta gag gct cct tcc gaa caagca ccg act gag caa agg cct ggg 2169 Ser Leu Glu Ala Pro Ser Glu Gln AlaPro Thr Glu Gln Arg Pro Gly 695 700 705 gtg cag gag tgc tac cat ggt aatgga cag agt tat cga ggc aca tac 2217 Val Gln Glu Cys Tyr His Gly Asn GlyGln Ser Tyr Arg Gly Thr Tyr 710 715 720 tcc acc act gtc aca gga aga acctgc caa gct tgg tca tct atg aca 2265 Ser Thr Thr Val Thr Gly Arg Thr CysGln Ala Trp Ser Ser Met Thr 725 730 735 740 cca cac tcg cat agt cgg acccca gaa tac tac cca aat gct ggc ttg 2313 Pro His Ser His Ser Arg Thr ProGlu Tyr Tyr Pro Asn Ala Gly Leu 745 750 755 atc atg aac tac tgc agg aatcca gat gct gtg gca gct cct tat tgt 2361 Ile Met Asn Tyr Cys Arg Asn ProAsp Ala Val Ala Ala Pro Tyr Cys 760 765 770 tat acg agg gat ccc ggt gtcagg tgg gag tac tgc aac ctg acg caa 2409 Tyr Thr Arg Asp Pro Gly Val ArgTrp Glu Tyr Cys Asn Leu Thr Gln 775 780 785 tgc tca gac gca gaa ggg actgcc gtc gcg cct ccg act gtt acc ccg 2457 Cys Ser Asp Ala Glu Gly Thr AlaVal Ala Pro Pro Thr Val Thr Pro 790 795 800 gtt cca agc cta gag gct ccttcc gaa caa gca ccg act gag caa agg 2505 Val Pro Ser Leu Glu Ala Pro SerGlu Gln Ala Pro Thr Glu Gln Arg 805 810 815 820 cct ggg gtg cag gag tgctac cat ggt aat gga cag agt tat cga ggc 2553 Pro Gly Val Gln Glu Cys TyrHis Gly Asn Gly Gln Ser Tyr Arg Gly 825 830 835 aca tac tcc acc act gtcaca gga aga acc tgc caa gct tgg tca tct 2601 Thr Tyr Ser Thr Thr Val ThrGly Arg Thr Cys Gln Ala Trp Ser Ser 840 845 850 atg aca cca cac tcg catagt cgg acc cca gaa tac tac cca aat gct 2649 Met Thr Pro His Ser His SerArg Thr Pro Glu Tyr Tyr Pro Asn Ala 855 860 865 ggc ttg atc atg aac tactgc agg aat cca gat gct gtg gca gct cct 2697 Gly Leu Ile Met Asn Tyr CysArg Asn Pro Asp Ala Val Ala Ala Pro 870 875 880 tat tgt tat acg agg gatccc ggt gtc agg tgg gag tac tgc aac ctg 2745 Tyr Cys Tyr Thr Arg Asp ProGly Val Arg Trp Glu Tyr Cys Asn Leu 885 890 895 900 acg caa tgc tca gacgca gaa ggg act gcc gtc gcg cct ccg act gtt 2793 Thr Gln Cys Ser Asp AlaGlu Gly Thr Ala Val Ala Pro Pro Thr Val 905 910 915 acc ccg gtt cca agccta gag gct cct tcc gaa caa gca ccg act gag 2841 Thr Pro Val Pro Ser LeuGlu Ala Pro Ser Glu Gln Ala Pro Thr Glu 920 925 930 caa agg cct ggg gtgcag gag tgc tac cat ggt aat gga cag agt tat 2889 Gln Arg Pro Gly Val GlnGlu Cys Tyr His Gly Asn Gly Gln Ser Tyr 935 940 945 cga ggc aca tac tccacc act gtc aca gga aga acc tgc caa gct tgg 2937 Arg Gly Thr Tyr Ser ThrThr Val Thr Gly Arg Thr Cys Gln Ala Trp 950 955 960 tca tct atg aca ccacac tcg cat agt cgg acc cca gaa tac tac cca 2985 Ser Ser Met Thr Pro HisSer His Ser Arg Thr Pro Glu Tyr Tyr Pro 965 970 975 980 aat gct ggc ttgatc atg aac tac tgc agg aat cca gat gct gtg gca 3033 Asn Ala Gly Leu IleMet Asn Tyr Cys Arg Asn Pro Asp Ala Val Ala 985 990 995 gct cct tat tgttat acg agg gat ccc ggt gtc agg tgg gag tac tgc 3081 Ala Pro Tyr Cys TyrThr Arg Asp Pro Gly Val Arg Trp Glu Tyr Cys 1000 1005 1010 aac ctg acgcaa tgc tca gac gca gaa ggg act gcc gtc gcg cct ccg 3129 Asn Leu Thr GlnCys Ser Asp Ala Glu Gly Thr Ala Val Ala Pro Pro 1015 1020 1025 act gttacc ccg gtt cca agc cta gag gct cct tcc gaa caa gca ccg 3177 Thr Val ThrPro Val Pro Ser Leu Glu Ala Pro Ser Glu Gln Ala Pro 1030 1035 1040 actgag caa agg cct ggg gtg cag gag tgc tac cat ggt aat gga cag 3225 Thr GluGln Arg Pro Gly Val Gln Glu Cys Tyr His Gly Asn Gly Gln 1045 1050 10551060 agt tat cga ggc aca tac tcc acc act gtc aca gga aga acc tgc caa3273 Ser Tyr Arg Gly Thr Tyr Ser Thr Thr Val Thr Gly Arg Thr Cys Gln1065 1070 1075 gct tgg tca tct atg aca cca cac tcg cat agt cgg acc ccagaa tac 3321 Ala Trp Ser Ser Met Thr Pro His Ser His Ser Arg Thr Pro GluTyr 1080 1085 1090 tac cca aat gct ggc ttg atc atg aac tac tgc agg aatcca gat gct 3369 Tyr Pro Asn Ala Gly Leu Ile Met Asn Tyr Cys Arg Asn ProAsp Ala 1095 1100 1105 gtg gca gct cct tat tgt tat acg agg gat ccc ggtgtc agg tgg gag 3417 Val Ala Ala Pro Tyr Cys Tyr Thr Arg Asp Pro Gly ValArg Trp Glu 1110 1115 1120 tac tgc aac ctg acg caa tgc tca gac gca gaaggg act gcc gtc gcg 3465 Tyr Cys Asn Leu Thr Gln Cys Ser Asp Ala Glu GlyThr Ala Val Ala 1125 1130 1135 1140 cct ccg act gtt acc ccg gtt cca agccta gag gct cct tcc gaa caa 3513 Pro Pro Thr Val Thr Pro Val Pro Ser LeuGlu Ala Pro Ser Glu Gln 1145 1150 1155 gca ccg act gag caa agg cct ggggtg cag gag tgc tac cat ggt aat 3561 Ala Pro Thr Glu Gln Arg Pro Gly ValGln Glu Cys Tyr His Gly Asn 1160 1165 1170 gga cag agt tat cga ggc acatac tcc acc act gtc aca gga aga acc 3609 Gly Gln Ser Tyr Arg Gly Thr TyrSer Thr Thr Val Thr Gly Arg Thr 1175 1180 1185 tgc caa gct tgg tca tctatg aca cca cac tcg cat agt cgg acc cca 3657 Cys Gln Ala Trp Ser Ser MetThr Pro His Ser His Ser Arg Thr Pro 1190 1195 1200 gaa tac tac cca aatgct ggc ttg atc atg aac tac tgc agg aat cca 3705 Glu Tyr Tyr Pro Asn AlaGly Leu Ile Met Asn Tyr Cys Arg Asn Pro 1205 1210 1215 1220 gat gct gtggca gct cct tat tgt tat acg agg gat ccc ggt gtc agg 3753 Asp Ala Val AlaAla Pro Tyr Cys Tyr Thr Arg Asp Pro Gly Val Arg 1225 1230 1235 tgg gagtac tgc aac ctg acg caa tgc tca gac gca gaa ggg act gcc 3801 Trp Glu TyrCys Asn Leu Thr Gln Cys Ser Asp Ala Glu Gly Thr Ala 1240 1245 1250 gtcgcg cct ccg act gtt acc ccg gtt cca agc cta gag gct cct tcc 3849 Val AlaPro Pro Thr Val Thr Pro Val Pro Ser Leu Glu Ala Pro Ser 1255 1260 1265gaa caa gca ccg act gag caa agg cct ggg gtg cag gag tgc tac cat 3897 GluGln Ala Pro Thr Glu Gln Arg Pro Gly Val Gln Glu Cys Tyr His 1270 12751280 ggt aat gga cag agt tat cga ggc aca tac tcc acc act gtc aca gga3945 Gly Asn Gly Gln Ser Tyr Arg Gly Thr Tyr Ser Thr Thr Val Thr Gly1285 1290 1295 1300 aga acc tgc caa gct tgg tca tct atg aca cca cac tcgcat agt cgg 3993 Arg Thr Cys Gln Ala Trp Ser Ser Met Thr Pro His Ser HisSer Arg 1305 1310 1315 acc cca gaa tac tac cca aat gct ggc ttg atc atgaac tac tgc agg 4041 Thr Pro Glu Tyr Tyr Pro Asn Ala Gly Leu Ile Met AsnTyr Cys Arg 1320 1325 1330 aat cca gat gct gtg gca gct cct tat tgt tatacg agg gat ccc ggt 4089 Asn Pro Asp Ala Val Ala Ala Pro Tyr Cys Tyr ThrArg Asp Pro Gly 1335 1340 1345 gtc agg tgg gag tac tgc aac ctg acg caatgc tca gac gca gaa ggg 4137 Val Arg Trp Glu Tyr Cys Asn Leu Thr Gln CysSer Asp Ala Glu Gly 1350 1355 1360 act gcc gtc gcg cct ccg act gtt accccg gtt cca agc cta gag gct 4185 Thr Ala Val Ala Pro Pro Thr Val Thr ProVal Pro Ser Leu Glu Ala 1365 1370 1375 1380 cct tcc gaa caa gca ccg actgag caa agg cct ggg gtg cag gag tgc 4233 Pro Ser Glu Gln Ala Pro Thr GluGln Arg Pro Gly Val Gln Glu Cys 1385 1390 1395 tac cat ggt aat gga cagagt tat cga ggc aca tac tcc acc act gtc 4281 Tyr His Gly Asn Gly Gln SerTyr Arg Gly Thr Tyr Ser Thr Thr Val 1400 1405 1410 aca gga aga acc tgccaa gct tgg tca tct atg aca cca cac tcg cat 4329 Thr Gly Arg Thr Cys GlnAla Trp Ser Ser Met Thr Pro His Ser His 1415 1420 1425 agt cgg acc ccagaa tac tac cca aat gct ggc ttg atc atg aac tac 4377 Ser Arg Thr Pro GluTyr Tyr Pro Asn Ala Gly Leu Ile Met Asn Tyr 1430 1435 1440 tgc agg aatcca gat gct gtg gca gct cct tat tgt tat acg agg gat 4425 Cys Arg Asn ProAsp Ala Val Ala Ala Pro Tyr Cys Tyr Thr Arg Asp 1445 1450 1455 1460 cccggt gtc agg tgg gag tac tgc aac ctg acg caa tgc tca gac gca 4473 Pro GlyVal Arg Trp Glu Tyr Cys Asn Leu Thr Gln Cys Ser Asp Ala 1465 1470 1475gaa ggg act gcc gtc gcg cct ccg act gtt acc ccg gtt cca agc cta 4521 GluGly Thr Ala Val Ala Pro Pro Thr Val Thr Pro Val Pro Ser Leu 1480 14851490 gag gct cct tcc gaa caa gca ccg act gag caa agg cct ggg gtg cag4569 Glu Ala Pro Ser Glu Gln Ala Pro Thr Glu Gln Arg Pro Gly Val Gln1495 1500 1505 gag tgc tac cat ggt aat gga cag agt tat cga ggc aca tactcc acc 4617 Glu Cys Tyr His Gly Asn Gly Gln Ser Tyr Arg Gly Thr Tyr SerThr 1510 1515 1520 act gtc aca gga aga acc tgc caa gct tgg tca tct atgaca cca cac 4665 Thr Val Thr Gly Arg Thr Cys Gln Ala Trp Ser Ser Met ThrPro His 1525 1530 1535 1540 tcg cat agt cgg acc cca gaa tac tac cca aatgct ggc ttg atc atg 4713 Ser His Ser Arg Thr Pro Glu Tyr Tyr Pro Asn AlaGly Leu Ile Met 1545 1550 1555 aac tac tgc agg aat cca gat gct gtg gcagct cct tat tgt tat acg 4761 Asn Tyr Cys Arg Asn Pro Asp Ala Val Ala AlaPro Tyr Cys Tyr Thr 1560 1565 1570 agg gat ccc ggt gtc agg tgg gag tactgc aac ctg acg caa tgc tca 4809 Arg Asp Pro Gly Val Arg Trp Glu Tyr CysAsn Leu Thr Gln Cys Ser 1575 1580 1585 gac gca gaa ggg act gcc gtc gcgcct ccg act gtt acc ccg gtt cca 4857 Asp Ala Glu Gly Thr Ala Val Ala ProPro Thr Val Thr Pro Val Pro 1590 1595 1600 agc cta gag gct cct tcc gaacaa gca ccg act gag caa agg cct ggg 4905 Ser Leu Glu Ala Pro Ser Glu GlnAla Pro Thr Glu Gln Arg Pro Gly 1605 1610 1615 1620 gtg cag gag tgc taccat ggt aat gga cag agt tat cga ggc aca tac 4953 Val Gln Glu Cys Tyr HisGly Asn Gly Gln Ser Tyr Arg Gly Thr Tyr 1625 1630 1635 tcc acc act gtcaca gga aga acc tgc caa gct tgg tca tct atg aca 5001 Ser Thr Thr Val ThrGly Arg Thr Cys Gln Ala Trp Ser Ser Met Thr 1640 1645 1650 cca cac tcgcat agt cgg acc cca gaa tac tac cca aat gct ggc ttg 5049 Pro His Ser HisSer Arg Thr Pro Glu Tyr Tyr Pro Asn Ala Gly Leu 1655 1660 1665 atc atgaac tac tgc agg aat cca gat gct gtg gca gct cct tat tgt 5097 Ile Met AsnTyr Cys Arg Asn Pro Asp Ala Val Ala Ala Pro Tyr Cys 1670 1675 1680 tatacg agg gat ccc ggt gtc agg tgg gag tac tgc aac ctg acg caa 5145 Tyr ThrArg Asp Pro Gly Val Arg Trp Glu Tyr Cys Asn Leu Thr Gln 1685 1690 16951700 tgc tca gac gca gaa ggg act gcc gtc gcg cct ccg act gtt acc ccg5193 Cys Ser Asp Ala Glu Gly Thr Ala Val Ala Pro Pro Thr Val Thr Pro1705 1710 1715 gtt cca agc cta gag gct cct tcc gaa caa gca ccg act gagcaa agg 5241 Val Pro Ser Leu Glu Ala Pro Ser Glu Gln Ala Pro Thr Glu GlnArg 1720 1725 1730 cct ggg gtg cag gag tgc tac cat ggt aat gga cag agttat cga ggc 5289 Pro Gly Val Gln Glu Cys Tyr His Gly Asn Gly Gln Ser TyrArg Gly 1735 1740 1745 aca tac tcc acc act gtc aca gga aga acc tgc caagct tgg tca tct 5337 Thr Tyr Ser Thr Thr Val Thr Gly Arg Thr Cys Gln AlaTrp Ser Ser 1750 1755 1760 atg aca cca cac tcg cat agt cgg acc cca gaatac tac cca aat gct 5385 Met Thr Pro His Ser His Ser Arg Thr Pro Glu TyrTyr Pro Asn Ala 1765 1770 1775 1780 ggc ttg atc atg aac tac tgc agg aatcca gat gct gtg gca gct cct 5433 Gly Leu Ile Met Asn Tyr Cys Arg Asn ProAsp Ala Val Ala Ala Pro 1785 1790 1795 tat tgt tat acg agg gat ccc ggtgtc agg tgg gag tac tgc aac ctg 5481 Tyr Cys Tyr Thr Arg Asp Pro Gly ValArg Trp Glu Tyr Cys Asn Leu 1800 1805 1810 acg caa tgc tca gac gca gaaggg act gcc gtc gcg cct ccg act gtt 5529 Thr Gln Cys Ser Asp Ala Glu GlyThr Ala Val Ala Pro Pro Thr Val 1815 1820 1825 acc ccg gtt cca agc ctagag gct cct tcc gaa caa gca ccg act gag 5577 Thr Pro Val Pro Ser Leu GluAla Pro Ser Glu Gln Ala Pro Thr Glu 1830 1835 1840 caa agg cct ggg gtgcag gag tgc tac cat ggt aat gga cag agt tat 5625 Gln Arg Pro Gly Val GlnGlu Cys Tyr His Gly Asn Gly Gln Ser Tyr 1845 1850 1855 1860 cga ggc acatac tcc acc act gtc aca gga aga acc tgc caa gct tgg 5673 Arg Gly Thr TyrSer Thr Thr Val Thr Gly Arg Thr Cys Gln Ala Trp 1865 1870 1875 tca tctatg aca cca cac tcg cat agt cgg acc cca gaa tac tac cca 5721 Ser Ser MetThr Pro His Ser His Ser Arg Thr Pro Glu Tyr Tyr Pro 1880 1885 1890 aatgct ggc ttg atc atg aac tac tgc agg aat cca gat gct gtg gca 5769 Asn AlaGly Leu Ile Met Asn Tyr Cys Arg Asn Pro Asp Ala Val Ala 1895 1900 1905gct cct tat tgt tat acg agg gat ccc ggt gtc agg tgg gag tac tgc 5817 AlaPro Tyr Cys Tyr Thr Arg Asp Pro Gly Val Arg Trp Glu Tyr Cys 1910 19151920 aac ctg acg caa tgc tca gac gca gaa ggg act gcc gtc gcg cct ccg5865 Asn Leu Thr Gln Cys Ser Asp Ala Glu Gly Thr Ala Val Ala Pro Pro1925 1930 1935 1940 act gtt acc ccg gtt cca agc cta gag gct cct tcc gaacaa gca ccg 5913 Thr Val Thr Pro Val Pro Ser Leu Glu Ala Pro Ser Glu GlnAla Pro 1945 1950 1955 act gag caa agg cct ggg gtg cag gag tgc tac catggt aat gga cag 5961 Thr Glu Gln Arg Pro Gly Val Gln Glu Cys Tyr His GlyAsn Gly Gln 1960 1965 1970 agt tat cga ggc aca tac tcc acc act gtc acagga aga acc tgc caa 6009 Ser Tyr Arg Gly Thr Tyr Ser Thr Thr Val Thr GlyArg Thr Cys Gln 1975 1980 1985 gct tgg tca tct atg aca cca cac tcg catagt cgg acc cca gaa tac 6057 Ala Trp Ser Ser Met Thr Pro His Ser His SerArg Thr Pro Glu Tyr 1990 1995 2000 tac cca aat gct ggc ttg atc atg aactac tgc agg aat cca gat gct 6105 Tyr Pro Asn Ala Gly Leu Ile Met Asn TyrCys Arg Asn Pro Asp Ala 2005 2010 2015 2020 gtg gca gct cct tat tgt tatacg agg gat ccc ggt gtc agg tgg gag 6153 Val Ala Ala Pro Tyr Cys Tyr ThrArg Asp Pro Gly Val Arg Trp Glu 2025 2030 2035 tac tgc aac ctg acg caatgc tca gac gca gaa ggg act gcc gtc gcg 6201 Tyr Cys Asn Leu Thr Gln CysSer Asp Ala Glu Gly Thr Ala Val Ala 2040 2045 2050 cct ccg act gtt accccg gtt cca agc cta gag gct cct tcc gaa caa 6249 Pro Pro Thr Val Thr ProVal Pro Ser Leu Glu Ala Pro Ser Glu Gln 2055 2060 2065 gca ccg act gagcaa agg cct ggg gtg cag gag tgc tac cat ggt aat 6297 Ala Pro Thr Glu GlnArg Pro Gly Val Gln Glu Cys Tyr His Gly Asn 2070 2075 2080 gga cag agttat cga ggc aca tac tcc acc act gtc aca gga aga acc 6345 Gly Gln Ser TyrArg Gly Thr Tyr Ser Thr Thr Val Thr Gly Arg Thr 2085 2090 2095 2100 tgccaa gct tgg tca tct atg aca cca cac tcg cat agt cgg acc cca 6393 Cys GlnAla Trp Ser Ser Met Thr Pro His Ser His Ser Arg Thr Pro 2105 2110 2115gaa tac tac cca aat gct ggc ttg atc atg aac tac tgc agg aat cca 6441 GluTyr Tyr Pro Asn Ala Gly Leu Ile Met Asn Tyr Cys Arg Asn Pro 2120 21252130 gat gct gtg gca gct cct tat tgt tat acg agg gat ccc ggt gtc agg6489 Asp Ala Val Ala Ala Pro Tyr Cys Tyr Thr Arg Asp Pro Gly Val Arg2135 2140 2145 tgg gag tac tgc aac ctg acg caa tgc tca gac gca gaa gggact gcc 6537 Trp Glu Tyr Cys Asn Leu Thr Gln Cys Ser Asp Ala Glu Gly ThrAla 2150 2155 2160 gtc gcg cct ccg act gtt acc ccg gtt cca agc cta gaggct cct tcc 6585 Val Ala Pro Pro Thr Val Thr Pro Val Pro Ser Leu Glu AlaPro Ser 2165 2170 2175 2180 gaa caa gca ccg act gag caa agg cct ggg gtgcag gag tgc tac cat 6633 Glu Gln Ala Pro Thr Glu Gln Arg Pro Gly Val GlnGlu Cys Tyr His 2185 2190 2195 ggt aat gga cag agt tat cga ggc aca tactcc acc act gtc aca gga 6681 Gly Asn Gly Gln Ser Tyr Arg Gly Thr Tyr SerThr Thr Val Thr Gly 2200 2205 2210 aga acc tgc caa gct tgg tca tct atgaca cca cac tcg cat agt cgg 6729 Arg Thr Cys Gln Ala Trp Ser Ser Met ThrPro His Ser His Ser Arg 2215 2220 2225 acc cca gaa tac tac cca aat gctggc ttg atc atg aac tac tgc agg 6777 Thr Pro Glu Tyr Tyr Pro Asn Ala GlyLeu Ile Met Asn Tyr Cys Arg 2230 2235 2240 aat cca gat gct gtg gca gctcct tat tgt tat acg agg gat ccc ggt 6825 Asn Pro Asp Ala Val Ala Ala ProTyr Cys Tyr Thr Arg Asp Pro Gly 2245 2250 2255 2260 gtc agg tgg gag tactgc aac ctg acg caa tgc tca gac gca gaa ggg 6873 Val Arg Trp Glu Tyr CysAsn Leu Thr Gln Cys Ser Asp Ala Glu Gly 2265 2270 2275 act gcc gtc gcgcct ccg act gtt acc ccg gtt cca agc cta gag gct 6921 Thr Ala Val Ala ProPro Thr Val Thr Pro Val Pro Ser Leu Glu Ala 2280 2285 2290 cct tcc gaacaa gca ccg act gag caa agg cct ggg gtg cag gag tgc 6969 Pro Ser Glu GlnAla Pro Thr Glu Gln Arg Pro Gly Val Gln Glu Cys 2295 2300 2305 tac catggt aat gga cag agt tat cga ggc aca tac tcc acc act gtc 7017 Tyr His GlyAsn Gly Gln Ser Tyr Arg Gly Thr Tyr Ser Thr Thr Val 2310 2315 2320 acagga aga acc tgc caa gct tgg tca tct atg aca cca cac tcg cat 7065 Thr GlyArg Thr Cys Gln Ala Trp Ser Ser Met Thr Pro His Ser His 2325 2330 23352340 agt cgg acc cca gaa tac tac cca aat gct ggc ttg atc atg aac tac7113 Ser Arg Thr Pro Glu Tyr Tyr Pro Asn Ala Gly Leu Ile Met Asn Tyr2345 2350 2355 tgc agg aat cca gat gct gtg gca gct cct tat tgt tat acgagg gat 7161 Cys Arg Asn Pro Asp Ala Val Ala Ala Pro Tyr Cys Tyr Thr ArgAsp 2360 2365 2370 ccc ggt gtc agg tgg gag tac tgc aac ctg acg caa tgctca gac gca 7209 Pro Gly Val Arg Trp Glu Tyr Cys Asn Leu Thr Gln Cys SerAsp Ala 2375 2380 2385 gaa ggg act gcc gtc gcg cct ccg act gtt acc ccggtt cca agc cta 7257 Glu Gly Thr Ala Val Ala Pro Pro Thr Val Thr Pro ValPro Ser Leu 2390 2395 2400 gag gct cct tcc gaa caa gca ccg act gag caaagg cct ggg gtg cag 7305 Glu Ala Pro Ser Glu Gln Ala Pro Thr Glu Gln ArgPro Gly Val Gln 2405 2410 2415 2420 gag tgc tac cat ggt aat gga cag agttat cga ggc aca tac tcc acc 7353 Glu Cys Tyr His Gly Asn Gly Gln Ser TyrArg Gly Thr Tyr Ser Thr 2425 2430 2435 act gtc aca gga aga acc tgc caagct tgg tca tct atg aca cca cac 7401 Thr Val Thr Gly Arg Thr Cys Gln AlaTrp Ser Ser Met Thr Pro His 2440 2445 2450 tcg cat agt cgg acc cca gaatac tac cca aat gct ggc ttg atc atg 7449 Ser His Ser Arg Thr Pro Glu TyrTyr Pro Asn Ala Gly Leu Ile Met 2455 2460 2465 aac tac tgc agg aat ccagat gct gtg gca gct cct tat tgt tat acg 7497 Asn Tyr Cys Arg Asn Pro AspAla Val Ala Ala Pro Tyr Cys Tyr Thr 2470 2475 2480 agg gat ccc ggt gtcagg tgg gag tac tgc aac ctg acg caa tgc tca 7545 Arg Asp Pro Gly Val ArgTrp Glu Tyr Cys Asn Leu Thr Gln Cys Ser 2485 2490 2495 2500 gac gca gaaggg act gcc gtc gcg cct ccg act gtt acc ccg gtt cca 7593 Asp Ala Glu GlyThr Ala Val Ala Pro Pro Thr Val Thr Pro Val Pro 2505 2510 2515 agc ctagag gct cct tcc gaa caa gca ccg act gag caa agg cct ggg 7641 Ser Leu GluAla Pro Ser Glu Gln Ala Pro Thr Glu Gln Arg Pro Gly 2520 2525 2530 gtgcag gag tgc tac cat ggt aat gga cag agt tat cga ggc aca tac 7689 Val GlnGlu Cys Tyr His Gly Asn Gly Gln Ser Tyr Arg Gly Thr Tyr 2535 2540 2545tcc acc act gtc aca gga aga acc tgc caa gct tgg tca tct atg aca 7737 SerThr Thr Val Thr Gly Arg Thr Cys Gln Ala Trp Ser Ser Met Thr 2550 25552560 cca cac tcg cat agt cgg acc cca gaa tac tac cca aat gct ggc ttg7785 Pro His Ser His Ser Arg Thr Pro Glu Tyr Tyr Pro Asn Ala Gly Leu2565 2570 2575 2580 atc atg aac tac tgc agg aat cca gat gct gtg gca gctcct tat tgt 7833 Ile Met Asn Tyr Cys Arg Asn Pro Asp Ala Val Ala Ala ProTyr Cys 2585 2590 2595 tat acg agg gat ccc ggt gtc agg tgg gag tac tgcaac ctg acg caa 7881 Tyr Thr Arg Asp Pro Gly Val Arg Trp Glu Tyr Cys AsnLeu Thr Gln 2600 2605 2610 tgc tca gac gca gaa ggg act gcc gtc gcg cctccg act gtt acc ccg 7929 Cys Ser Asp Ala Glu Gly Thr Ala Val Ala Pro ProThr Val Thr Pro 2615 2620 2625 gtt cca agc cta gag gct cct tcc gaa caagca ccg act gag cag agg 7977 Val Pro Ser Leu Glu Ala Pro Ser Glu Gln AlaPro Thr Glu Gln Arg 2630 2635 2640 cct ggg gtg cag gag tgc tac cac ggtaat gga cag agt tat cga ggc 8025 Pro Gly Val Gln Glu Cys Tyr His Gly AsnGly Gln Ser Tyr Arg Gly 2645 2650 2655 2660 aca tac tcc acc act gtc actgga aga acc tgc caa gct tgg tca tct 8073 Thr Tyr Ser Thr Thr Val Thr GlyArg Thr Cys Gln Ala Trp Ser Ser 2665 2670 2675 atg aca cca cac tcg catagt cgg acc cca gaa tac tac cca aat gct 8121 Met Thr Pro His Ser His SerArg Thr Pro Glu Tyr Tyr Pro Asn Ala 2680 2685 2690 ggc ttg atc atg aactac tgc agg aat cca gat gct gtg gca gct cct 8169 Gly Leu Ile Met Asn TyrCys Arg Asn Pro Asp Ala Val Ala Ala Pro 2695 2700 2705 tat tgt tat acgagg gat ccc ggt gtc agg tgg gag tac tgc aac ctg 8217 Tyr Cys Tyr Thr ArgAsp Pro Gly Val Arg Trp Glu Tyr Cys Asn Leu 2710 2715 2720 acg caa tgctca gac gca gaa ggg act gcc gtc gcg cct ccg act gtt 8265 Thr Gln Cys SerAsp Ala Glu Gly Thr Ala Val Ala Pro Pro Thr Val 2725 2730 2735 2740 accccg gtt cca agc cta gag gct cct tcc gaa caa gca ccg act gag 8313 Thr ProVal Pro Ser Leu Glu Ala Pro Ser Glu Gln Ala Pro Thr Glu 2745 2750 2755caa agg cct ggg gtg cag gag tgc tac cat ggt aat gga cag agt tat 8361 GlnArg Pro Gly Val Gln Glu Cys Tyr His Gly Asn Gly Gln Ser Tyr 2760 27652770 cga ggc aca tac tcc acc act gtc aca gga aga acc tgc caa gct tgg8409 Arg Gly Thr Tyr Ser Thr Thr Val Thr Gly Arg Thr Cys Gln Ala Trp2775 2780 2785 tca tct atg aca cca cac tcg cat agt cgg acc cca gaa tactac cca 8457 Ser Ser Met Thr Pro His Ser His Ser Arg Thr Pro Glu Tyr TyrPro 2790 2795 2800 aat gct ggc ttg atc atg aac tac tgc agg aat cca gatgct gtg gca 8505 Asn Ala Gly Leu Ile Met Asn Tyr Cys Arg Asn Pro Asp AlaVal Ala 2805 2810 2815 2820 gct cct tat tgt tat acg agg gat ccc ggt gtcagg tgg gag tac tgc 8553 Ala Pro Tyr Cys Tyr Thr Arg Asp Pro Gly Val ArgTrp Glu Tyr Cys 2825 2830 2835 aac ctg acg caa tgc tca gac gca gaa gggact gcc gtc gcg cct ccg 8601 Asn Leu Thr Gln Cys Ser Asp Ala Glu Gly ThrAla Val Ala Pro Pro 2840 2845 2850 act gtt acc ccg gtt cca agc cta gaggct cct tcc gaa caa gca ccg 8649 Thr Val Thr Pro Val Pro Ser Leu Glu AlaPro Ser Glu Gln Ala Pro 2855 2860 2865 act gag caa agg cct ggg gtg caggag tgc tac cat ggt aat gga cag 8697 Thr Glu Gln Arg Pro Gly Val Gln GluCys Tyr His Gly Asn Gly Gln 2870 2875 2880 agt tat cga ggc aca tac tccacc act gtc aca gga aga acc tgc caa 8745 Ser Tyr Arg Gly Thr Tyr Ser ThrThr Val Thr Gly Arg Thr Cys Gln 2885 2890 2895 2900 gct tgg tca tct atgaca cca cac tcg cat agt cgg acc cca gaa tac 8793 Ala Trp Ser Ser Met ThrPro His Ser His Ser Arg Thr Pro Glu Tyr 2905 2910 2915 tac cca aat gctggc ttg atc atg aac tac tgc agg aat cca gat gct 8841 Tyr Pro Asn Ala GlyLeu Ile Met Asn Tyr Cys Arg Asn Pro Asp Ala 2920 2925 2930 gtg gca gctcct tat tgt tat acg agg gat ccc ggt gtc agg tgg gag 8889 Val Ala Ala ProTyr Cys Tyr Thr Arg Asp Pro Gly Val Arg Trp Glu 2935 2940 2945 tac tgcaac ctg acg caa tgc tca gac gca gaa ggg act gcc gtc gcg 8937 Tyr Cys AsnLeu Thr Gln Cys Ser Asp Ala Glu Gly Thr Ala Val Ala 2950 2955 2960 cctccg act gtt acc ccg gtt cca agc cta gag gct cct tcc gaa caa 8985 Pro ProThr Val Thr Pro Val Pro Ser Leu Glu Ala Pro Ser Glu Gln 2965 2970 29752980 gca ccg act gag cag agg cct ggg gtg cag gag tgc tac cac ggt aat9033 Ala Pro Thr Glu Gln Arg Pro Gly Val Gln Glu Cys Tyr His Gly Asn2985 2990 2995 gga cag agt tat cga ggc aca tac tcc acc act gtc act ggaaga acc 9081 Gly Gln Ser Tyr Arg Gly Thr Tyr Ser Thr Thr Val Thr Gly ArgThr 3000 3005 3010 tgc caa gct tgg tca tct atg aca cca cac tcg cat agtcgg acc cca 9129 Cys Gln Ala Trp Ser Ser Met Thr Pro His Ser His Ser ArgThr Pro 3015 3020 3025 gaa tac tac cca aat gct ggc ttg atc atg aac tactgc agg aat cca 9177 Glu Tyr Tyr Pro Asn Ala Gly Leu Ile Met Asn Tyr CysArg Asn Pro 3030 3035 3040 gat gct gtg gca gct cct tat tgt tat acg agggat ccc ggt gtc agg 9225 Asp Ala Val Ala Ala Pro Tyr Cys Tyr Thr Arg AspPro Gly Val Arg 3045 3050 3055 3060 tgg gag tac tgc aac ctg acg caa tgctca gac gca gaa ggg act gcc 9273 Trp Glu Tyr Cys Asn Leu Thr Gln Cys SerAsp Ala Glu Gly Thr Ala 3065 3070 3075 gtc gcg cct ccg act gtt acc ccggtt cca agc cta gag gct cct tcc 9321 Val Ala Pro Pro Thr Val Thr Pro ValPro Ser Leu Glu Ala Pro Ser 3080 3085 3090 gaa caa gca ccg act gag cagagg cct ggg gtg cag gag tgc tac cac 9369 Glu Gln Ala Pro Thr Glu Gln ArgPro Gly Val Gln Glu Cys Tyr His 3095 3100 3105 ggt aat gga cag agt tatcga ggc aca tac tcc acc act gtc act gga 9417 Gly Asn Gly Gln Ser Tyr ArgGly Thr Tyr Ser Thr Thr Val Thr Gly 3110 3115 3120 aga acc tgc caa gcttgg tca tct atg aca cca cac tcg cat agt cgg 9465 Arg Thr Cys Gln Ala TrpSer Ser Met Thr Pro His Ser His Ser Arg 3125 3130 3135 3140 acc cca gaatac tac cca aat gct ggc ttg atc atg aac tac tgc agg 9513 Thr Pro Glu TyrTyr Pro Asn Ala Gly Leu Ile Met Asn Tyr Cys Arg 3145 3150 3155 aat ccagat gct gtg gca gct cct tat tgt tat acg agg gat ccc ggt 9561 Asn Pro AspAla Val Ala Ala Pro Tyr Cys Tyr Thr Arg Asp Pro Gly 3160 3165 3170 gtcagg tgg gag tac tgc aac ctg acg caa tgc tca gac gca gaa ggg 9609 Val ArgTrp Glu Tyr Cys Asn Leu Thr Gln Cys Ser Asp Ala Glu Gly 3175 3180 3185act gcc gtc gcg cct ccg act gtt acc ccg gtt cca agc cta gag gct 9657 ThrAla Val Ala Pro Pro Thr Val Thr Pro Val Pro Ser Leu Glu Ala 3190 31953200 cct tcc gaa caa gca ccg act gag cag agg cct ggg gtg cag gag tgc9705 Pro Ser Glu Gln Ala Pro Thr Glu Gln Arg Pro Gly Val Gln Glu Cys3205 3210 3215 3220 tac cac ggt aat gga cag agt tat cga ggc aca tac tccacc act gtc 9753 Tyr His Gly Asn Gly Gln Ser Tyr Arg Gly Thr Tyr Ser ThrThr Val 3225 3230 3235 act gga aga acc tgc caa gct tgg tca tct atg acacca cac tcg cat 9801 Thr Gly Arg Thr Cys Gln Ala Trp Ser Ser Met Thr ProHis Ser His 3240 3245 3250 agt cgg acc cca gaa tac tac cca aat gct ggcttg atc atg aac tac 9849 Ser Arg Thr Pro Glu Tyr Tyr Pro Asn Ala Gly LeuIle Met Asn Tyr 3255 3260 3265 tgc agg aat cca gat gct gtg gca gct ccttat tgt tat acg agg gat 9897 Cys Arg Asn Pro Asp Ala Val Ala Ala Pro TyrCys Tyr Thr Arg Asp 3270 3275 3280 ccc ggt gtc agg tgg gag tac tgc aacctg acg caa tgc tca gac gca 9945 Pro Gly Val Arg Trp Glu Tyr Cys Asn LeuThr Gln Cys Ser Asp Ala 3285 3290 3295 3300 gaa ggg act gcc gtc gcg cctccg act gtt acc ccg gtt cca agc cta 9993 Glu Gly Thr Ala Val Ala Pro ProThr Val Thr Pro Val Pro Ser Leu 3305 3310 3315 gag gct cct tcc gaa caagca ccg act gag cag agg cct ggg gtg cag 10041 Glu Ala Pro Ser Glu GlnAla Pro Thr Glu Gln Arg Pro Gly Val Gln 3320 3325 3330 gag tgc tac cacggt aat gga cag agt tat cga ggc aca tac tcc acc 10089 Glu Cys Tyr HisGly Asn Gly Gln Ser Tyr Arg Gly Thr Tyr Ser Thr 3335 3340 3345 act gtcact gga aga acc tgc caa gct tgg tca tct atg aca cca cac 10137 Thr ValThr Gly Arg Thr Cys Gln Ala Trp Ser Ser Met Thr Pro His 3350 3355 3360tcg cat agt cgg acc cca gaa tac tac cca aat gct ggc ttg atc atg 10185Ser His Ser Arg Thr Pro Glu Tyr Tyr Pro Asn Ala Gly Leu Ile Met 33653370 3375 3380 aac tac tgc agg aat cca gat cct gtg gca gcc cct tat tgttat acg 10233 Asn Tyr Cys Arg Asn Pro Asp Pro Val Ala Ala Pro Tyr CysTyr Thr 3385 3390 3395 agg gat ccc agt gtc agg tgg gag tac tgc aac ctgaca caa tgc tca 10281 Arg Asp Pro Ser Val Arg Trp Glu Tyr Cys Asn LeuThr Gln Cys Ser 3400 3405 3410 gac gca gaa ggg act gcc gtc gcg cct ccaact att acc ccg att cca 10329 Asp Ala Glu Gly Thr Ala Val Ala Pro ProThr Ile Thr Pro Ile Pro 3415 3420 3425 agc cta gag gct cct tct gaa caagca cca act gag caa agg cct ggg 10377 Ser Leu Glu Ala Pro Ser Glu GlnAla Pro Thr Glu Gln Arg Pro Gly 3430 3435 3440 gtg cag gag tgc tac cacgga aat gga cag agt tat caa ggc aca tac 10425 Val Gln Glu Cys Tyr HisGly Asn Gly Gln Ser Tyr Gln Gly Thr Tyr 3445 3450 3455 3460 ttc att actgtc aca gga aga acc tgc caa gct tgg tca tct atg aca 10473 Phe Ile ThrVal Thr Gly Arg Thr Cys Gln Ala Trp Ser Ser Met Thr 3465 3470 3475 ccacac tcg cat agt cgg acc cca gca tac tac cca aat gct ggc ttg 10521 ProHis Ser His Ser Arg Thr Pro Ala Tyr Tyr Pro Asn Ala Gly Leu 3480 34853490 atc aag aac tac tgc cga aat cca gat cct gtg gca gcc cct tgg tgt10569 Ile Lys Asn Tyr Cys Arg Asn Pro Asp Pro Val Ala Ala Pro Trp Cys3495 3500 3505 tat aca aca gat ccc agt gtc agg tgg gag tac tgc aac ctgaca cga 10617 Tyr Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr Cys Asn LeuThr Arg 3510 3515 3520 tgc tca gat gca gaa tgg act gcc ttc gtc cct ccgaat gtt att ctg 10665 Cys Ser Asp Ala Glu Trp Thr Ala Phe Val Pro ProAsn Val Ile Leu 3525 3530 3535 3540 gct cca agc cta gag gct ttt ttt gaacaa gca ctg act gag gaa acc 10713 Ala Pro Ser Leu Glu Ala Phe Phe GluGln Ala Leu Thr Glu Glu Thr 3545 3550 3555 ccc ggg gta cag gac tgc tactac cat tat gga cag agt tac cga ggc 10761 Pro Gly Val Gln Asp Cys TyrTyr His Tyr Gly Gln Ser Tyr Arg Gly 3560 3565 3570 aca tac tcc acc actgtc aca gga aga act tgc caa gct tgg tca tct 10809 Thr Tyr Ser Thr ThrVal Thr Gly Arg Thr Cys Gln Ala Trp Ser Ser 3575 3580 3585 atg aca ccacac cag cat agt cgg acc cca gaa aac tac cca aat gct 10857 Met Thr ProHis Gln His Ser Arg Thr Pro Glu Asn Tyr Pro Asn Ala 3590 3595 3600 ggcctg acc agg aac tac tgc agg aat cca gat gct gag att cgc cct 10905 GlyLeu Thr Arg Asn Tyr Cys Arg Asn Pro Asp Ala Glu Ile Arg Pro 3605 36103615 3620 tgg tgt tac acc atg gat ccc agt gtc agg tgg gag tac tgc aacctg 10953 Trp Cys Tyr Thr Met Asp Pro Ser Val Arg Trp Glu Tyr Cys AsnLeu 3625 3630 3635 aca caa tgc ctg gtg aca gaa tca agt gtc ctt gca actctc acg gtg 11001 Thr Gln Cys Leu Val Thr Glu Ser Ser Val Leu Ala ThrLeu Thr Val 3640 3645 3650 gtc cca gat cca agc aca gag gct tct tct gaagaa gca cca acg gag 11049 Val Pro Asp Pro Ser Thr Glu Ala Ser Ser GluGlu Ala Pro Thr Glu 3655 3660 3665 caa agc ccc ggg gtc cag gat tgc taccat ggt gat gga cag agt tat 11097 Gln Ser Pro Gly Val Gln Asp Cys TyrHis Gly Asp Gly Gln Ser Tyr 3670 3675 3680 cga ggc tca ttc tct acc actgtc aca gga agg aca tgt cag tct tgg 11145 Arg Gly Ser Phe Ser Thr ThrVal Thr Gly Arg Thr Cys Gln Ser Trp 3685 3690 3695 3700 tcc tct atg acacca cac tgg cat cag agg aca aca gaa tat tat cca 11193 Ser Ser Met ThrPro His Trp His Gln Arg Thr Thr Glu Tyr Tyr Pro 3705 3710 3715 aat ggtggc ctg acc agg aac tac tgc agg aat cca gat gct gag att 11241 Asn GlyGly Leu Thr Arg Asn Tyr Cys Arg Asn Pro Asp Ala Glu Ile 3720 3725 3730agt cct tgg tgt tat acc atg gat ccc aat gtc aga tgg gag tac tgc 11289Ser Pro Trp Cys Tyr Thr Met Asp Pro Asn Val Arg Trp Glu Tyr Cys 37353740 3745 aac ctg aca caa tgt cca gtg aca gaa tca agt gtc ctt gcg acgtcc 11337 Asn Leu Thr Gln Cys Pro Val Thr Glu Ser Ser Val Leu Ala ThrSer 3750 3755 3760 acg gct gtt tct gaa caa gca cca acg gag caa agc cccaca gtc cag 11385 Thr Ala Val Ser Glu Gln Ala Pro Thr Glu Gln Ser ProThr Val Gln 3765 3770 3775 3780 gac tgc tac cat ggt gat gga cag agt tatcga ggc tca ttc tcc acc 11433 Asp Cys Tyr His Gly Asp Gly Gln Ser TyrArg Gly Ser Phe Ser Thr 3785 3790 3795 act gtt aca gga agg aca tgt cagtct tgg tcc tct atg aca cca cac 11481 Thr Val Thr Gly Arg Thr Cys GlnSer Trp Ser Ser Met Thr Pro His 3800 3805 3810 tgg cat cag aga acc acagaa tac tac cca aat ggt ggc ctg acc agg 11529 Trp His Gln Arg Thr ThrGlu Tyr Tyr Pro Asn Gly Gly Leu Thr Arg 3815 3820 3825 aac tac tgc aggaat cca gat gct gag att cgc cct tgg tgt tat acc 11577 Asn Tyr Cys ArgAsn Pro Asp Ala Glu Ile Arg Pro Trp Cys Tyr Thr 3830 3835 3840 atg gatccc agt gtc aga tgg gag tac tgc aac ctg acg caa tgt cca 11625 Met AspPro Ser Val Arg Trp Glu Tyr Cys Asn Leu Thr Gln Cys Pro 3845 3850 38553860 gtg atg gaa tca act ctc ctc aca act ccc acg gtg gtc cca gtt cca11673 Val Met Glu Ser Thr Leu Leu Thr Thr Pro Thr Val Val Pro Val Pro3865 3870 3875 agc aca gag ctt cct tct gaa gaa gca cca act gaa aac agcact ggg 11721 Ser Thr Glu Leu Pro Ser Glu Glu Ala Pro Thr Glu Asn SerThr Gly 3880 3885 3890 gtc cag gac tgc tac cga ggt gat gga cag agt tatcga ggc aca ctc 11769 Val Gln Asp Cys Tyr Arg Gly Asp Gly Gln Ser TyrArg Gly Thr Leu 3895 3900 3905 tcc acc act atc aca gga aga aca tgt cagtct tgg tcg tct atg aca 11817 Ser Thr Thr Ile Thr Gly Arg Thr Cys GlnSer Trp Ser Ser Met Thr 3910 3915 3920 cca cat tgg cat cgg agg atc ccatta tac tat cca aat gct ggc ctg 11865 Pro His Trp His Arg Arg Ile ProLeu Tyr Tyr Pro Asn Ala Gly Leu 3925 3930 3935 3940 acc agg aac tac tgcagg aat cca gat gct gag att cgc cct tgg tgt 11913 Thr Arg Asn Tyr CysArg Asn Pro Asp Ala Glu Ile Arg Pro Trp Cys 3945 3950 3955 tac acc atggat ccc agt gtc agg tgg gag tac tgc aac ctg aca cga 11961 Tyr Thr MetAsp Pro Ser Val Arg Trp Glu Tyr Cys Asn Leu Thr Arg 3960 3965 3970 tgtcca gtg aca gaa tcg agt gtc ctc aca act ccc aca gtg gcc ccg 12009 CysPro Val Thr Glu Ser Ser Val Leu Thr Thr Pro Thr Val Ala Pro 3975 39803985 gtt cca agc aca gag gct cct tct gaa caa gca cca cct gag aaa agc12057 Val Pro Ser Thr Glu Ala Pro Ser Glu Gln Ala Pro Pro Glu Lys Ser3990 3995 4000 cct gtg gtc cag gat tgc tac cat ggt gat gga cgg agt tatcga ggc 12105 Pro Val Val Gln Asp Cys Tyr His Gly Asp Gly Arg Ser TyrArg Gly 4005 4010 4015 4020 ata tcc tcc acc act gtc aca gga agg acc tgtcaa tct tgg tca tct 12153 Ile Ser Ser Thr Thr Val Thr Gly Arg Thr CysGln Ser Trp Ser Ser 4025 4030 4035 atg ata cca cac tgg cat cag agg acccca gaa aac tac cca aat gct 12201 Met Ile Pro His Trp His Gln Arg ThrPro Glu Asn Tyr Pro Asn Ala 4040 4045 4050 ggc ctg acc gag aac tac tgcagg aat cca gat tct ggg aaa caa ccc 12249 Gly Leu Thr Glu Asn Tyr CysArg Asn Pro Asp Ser Gly Lys Gln Pro 4055 4060 4065 tgg tgt tac aca accgat ccg tgt gtg agg tgg gag tac tgc aat ctg 12297 Trp Cys Tyr Thr ThrAsp Pro Cys Val Arg Trp Glu Tyr Cys Asn Leu 4070 4075 4080 aca caa tgctca gaa aca gaa tca ggt gtc cta gag act ccc act gtt 12345 Thr Gln CysSer Glu Thr Glu Ser Gly Val Leu Glu Thr Pro Thr Val 4085 4090 4095 4100gtt cca gtt cca agc atg gag gct cat tct gaa gca gca cca act gag 12393Val Pro Val Pro Ser Met Glu Ala His Ser Glu Ala Ala Pro Thr Glu 41054110 4115 caa acc cct gtg gtc cgg cag tgc tac cat ggt aat ggc cag agttat 12441 Gln Thr Pro Val Val Arg Gln Cys Tyr His Gly Asn Gly Gln SerTyr 4120 4125 4130 cga ggc aca ttc tcc acc act gtc aca gga agg aca tgtcaa tct tgg 12489 Arg Gly Thr Phe Ser Thr Thr Val Thr Gly Arg Thr CysGln Ser Trp 4135 4140 4145 tca tcc atg aca cca cac cgg cat cag agg acccca gaa aac tac cca 12537 Ser Ser Met Thr Pro His Arg His Gln Arg ThrPro Glu Asn Tyr Pro 4150 4155 4160 aat gat ggc ctg aca atg aac tac tgcagg aat cca gat gcc gat aca 12585 Asn Asp Gly Leu Thr Met Asn Tyr CysArg Asn Pro Asp Ala Asp Thr 4165 4170 4175 4180 ggc cct tgg tgt ttt accatg gac ccc agc atc agg tgg gag tac tgc 12633 Gly Pro Trp Cys Phe ThrMet Asp Pro Ser Ile Arg Trp Glu Tyr Cys 4185 4190 4195 aac ctg acg cgatgc tca gac aca gaa ggg act gtg gtc gct cct ccg 12681 Asn Leu Thr ArgCys Ser Asp Thr Glu Gly Thr Val Val Ala Pro Pro 4200 4205 4210 act gtcatc cag gtt cca agc cta ggg cct cct tct gaa caa gac tgt 12729 Thr ValIle Gln Val Pro Ser Leu Gly Pro Pro Ser Glu Gln Asp Cys 4215 4220 4225atg ttt ggg aat ggg aaa gga tac cgg ggc aag aag gca acc act gtt 12777Met Phe Gly Asn Gly Lys Gly Tyr Arg Gly Lys Lys Ala Thr Thr Val 42304235 4240 act ggg acg cca tgc cag gaa tgg gct gcc cag gag ccc cat agacac 12825 Thr Gly Thr Pro Cys Gln Glu Trp Ala Ala Gln Glu Pro His ArgHis 4245 4250 4255 4260 agc acg ttc att cca ggg aca aat aaa tgg gca ggtctg gaa aaa aat 12873 Ser Thr Phe Ile Pro Gly Thr Asn Lys Trp Ala GlyLeu Glu Lys Asn 4265 4270 4275 tac tgc cgt aac cct gat ggt gac atc aatggt ccc tgg tgc tac aca 12921 Tyr Cys Arg Asn Pro Asp Gly Asp Ile AsnGly Pro Trp Cys Tyr Thr 4280 4285 4290 atg aat cca aga aaa ctt ttt gactac tgt gat atc cct ctc tgt gca 12969 Met Asn Pro Arg Lys Leu Phe AspTyr Cys Asp Ile Pro Leu Cys Ala 4295 4300 4305 tcc tct tca ttt gat tgtggg aag cct caa gtg gag ccg aag aaa tgt 13017 Ser Ser Ser Phe Asp CysGly Lys Pro Gln Val Glu Pro Lys Lys Cys 4310 4315 4320 cct gga agc attgta ggg ggg tgt gtg gcc cac cca cat tcc tgg ccc 13065 Pro Gly Ser IleVal Gly Gly Cys Val Ala His Pro His Ser Trp Pro 4325 4330 4335 4340 tggcaa gtc agt ctc aga aca agg ttt gga aag cac ttc tgt gga ggc 13113 TrpGln Val Ser Leu Arg Thr Arg Phe Gly Lys His Phe Cys Gly Gly 4345 43504355 acc tta ata tcc cca gag tgg gtg ctg act gct gct cac tgc ttg aag13161 Thr Leu Ile Ser Pro Glu Trp Val Leu Thr Ala Ala His Cys Leu Lys4360 4365 4370 aag tcc tca agg cct tca tcc tac aag gtc atc ctg ggt gcacac caa 13209 Lys Ser Ser Arg Pro Ser Ser Tyr Lys Val Ile Leu Gly AlaHis Gln 4375 4380 4385 gaa gtg aac ctc gaa tct cat gtt cag gaa ata gaagtg tct agg ctg 13257 Glu Val Asn Leu Glu Ser His Val Gln Glu Ile GluVal Ser Arg Leu 4390 4395 4400 ttc ttg gag ccc aca caa gca gat att gccttg cta aag cta agc agg 13305 Phe Leu Glu Pro Thr Gln Ala Asp Ile AlaLeu Leu Lys Leu Ser Arg 4405 4410 4415 4420 cct gcc gtc atc act gac aaagta atg cca gct tgt ctg cca tcc cca 13353 Pro Ala Val Ile Thr Asp LysVal Met Pro Ala Cys Leu Pro Ser Pro 4425 4430 4435 gac tac atg gtc accgcc agg act gaa tgt tac atc act ggc tgg gga 13401 Asp Tyr Met Val ThrAla Arg Thr Glu Cys Tyr Ile Thr Gly Trp Gly 4440 4445 4450 gaa acc caaggt acc ttt ggg act ggc ctt ctc aag gaa gcc cag ctc 13449 Glu Thr GlnGly Thr Phe Gly Thr Gly Leu Leu Lys Glu Ala Gln Leu 4455 4460 4465 cttgtt att gag aat gaa gtg tgc aat cac tat aag tat att tgt gct 13497 LeuVal Ile Glu Asn Glu Val Cys Asn His Tyr Lys Tyr Ile Cys Ala 4470 44754480 gag cat ttg gcc aga ggc act gac agt tgc cag ggt gac agt gga ggg13545 Glu His Leu Ala Arg Gly Thr Asp Ser Cys Gln Gly Asp Ser Gly Gly4485 4490 4495 4500 cct ctg gtt tgc ttc gag aag gac aaa tac att tta caagga gtc act 13593 Pro Leu Val Cys Phe Glu Lys Asp Lys Tyr Ile Leu GlnGly Val Thr 4505 4510 4515 tct tgg ggt ctt ggc tgt gca cgc ccc aat aagcct ggt gtc tat gct 13641 Ser Trp Gly Leu Gly Cys Ala Arg Pro Asn LysPro Gly Val Tyr Ala 4520 4525 4530 cgt gtt tca agg ttt gtt act tgg attgag gga atg atg aga aat aat 13689 Arg Val Ser Arg Phe Val Thr Trp IleGlu Gly Met Met Arg Asn Asn 4535 4540 4545 taa ttggacggga gacagagtgaagcatcaacc tacttagaag ctgaaacgtg 13742 ggtaaggatt tagcatgctg gaaataatagacagcaatca aacgaagaca ctgttcccag 13802 ctaccagcta tgccaaacct tggcatttttggtatttttg tgtataagct tttaaggtct 13862 gactgacaaa ttctgtatta aggtgtcatagctatgacat ttgttaaaaa taaactctgc 13922 acttattttg atttga 13938 4 19 DNAArtificial Sequence PCR Primer 4 gaaggtgaag gtcggagtc 19 5 20 DNAArtificial Sequence PCR Primer 5 gaagatggtg atgggatttc 20 6 20 DNAArtificial Sequence PCR Probe 6 caagcttccc gttctcagcc 20 7 20 DNAArtificial Sequence Antisense Oligonucleotide 7 ggcaggtcct tcctgtgaca 208 20 DNA Artificial Sequence Antisense Oligonucleotide 8 tctgcgtctgagcattgcgt 20 9 20 DNA Artificial Sequence Antisense Oligonucleotide 9aagcttggca ggttcttcct 20 10 20 DNA Artificial Sequence AntisenseOligonucleotide 10 tcggaggcgc gacggcagtc 20 11 20 DNA ArtificialSequence Antisense Oligonucleotide 11 cggaggcgcg acggcagtcc 20 12 20 DNAArtificial Sequence Antisense Oligonucleotide 12 ggcaggttct tcctgtgaca20 13 20 DNA Artificial Sequence Antisense Oligonucleotide 13 ataacaataaggagctgcca 20 14 20 DNA Artificial Sequence Antisense Oligonucleotide 14gaccaagctt ggcaggttct 20 15 20 DNA Artificial Sequence AntisenseOligonucleotide 15 taacaataag gagctgccac 20 16 20 DNA ArtificialSequence Antisense Oligonucleotide 16 tgaccaagct tggcaggttc 20 17 20 DNAArtificial Sequence Antisense Oligonucleotide 17 ttctgcgtct gagcattgcg20 18 20 DNA Artificial Sequence Antisense Oligonucleotide 18 aacaataaggagctgccaca 20 19 20 DNA Artificial Sequence Antisense Oligonucleotide 19acctgacacc gggatccctc 20 20 20 DNA Artificial Sequence AntisenseOligonucleotide 20 ctgagcattg cgtcaggttg 20 21 20 DNA ArtificialSequence Antisense Oligonucleotide 21 agtagttcat gatcaagcca 20 22 20 DNAArtificial Sequence Antisense Oligonucleotide 22 gacggcagtc ccttctgcgt20 23 20 DNA Artificial Sequence Antisense Oligonucleotide 23 ggcaggttcttccagtgaca 20 24 20 DNA Artificial Sequence Antisense Oligonucleotide 24tgaccaagct tggcaagttc 20 25 20 DNA Artificial Sequence AntisenseOligonucleotide 25 tataacacca aggactaatc 20 26 20 DNA ArtificialSequence Antisense Oligonucleotide 26 ccatctgaca ttgggatcca 20 27 20 DNAArtificial Sequence Antisense Oligonucleotide 27 tgtggtgtca tagaggacca20 28 20 DNA Artificial Sequence Antisense Oligonucleotide 28 atgggatcctccgatgccaa 20 29 20 DNA Artificial Sequence Antisense Oligonucleotide 29acaccaaggg cgaatctcag 20 30 20 DNA Artificial Sequence AntisenseOligonucleotide 30 ttctgtcact ggacatcgtg 20 31 20 DNA ArtificialSequence Antisense Oligonucleotide 31 cacacggatc ggttgtgtaa 20 32 20 DNAArtificial Sequence Antisense Oligonucleotide 32 acatgtcctt cctgtgacag20 33 20 DNA Artificial Sequence Antisense Oligonucleotide 33 cagaaggaggccctaggctt 20 34 20 DNA Artificial Sequence Antisense Oligonucleotide 34ctggcggtga ccatgtagtc 20 35 20 DNA Artificial Sequence AntisenseOligonucleotide 35 tctaagtagg ttgatgcttc 20 36 20 DNA ArtificialSequence Antisense Oligonucleotide 36 tccttaccca cgtttcagct 20 37 20 DNAArtificial Sequence Antisense Oligonucleotide 37 ggaacagtgt cttcgtttga20 38 20 DNA Artificial Sequence Antisense Oligonucleotide 38 gtttggcatagctggtagct 20 39 20 DNA Artificial Sequence Antisense Oligonucleotide 39accttaaaag cttatacaca 20 40 20 DNA Artificial Sequence AntisenseOligonucleotide 40 atacagaatt tgtcagtcag 20 41 20 DNA ArtificialSequence Antisense Oligonucleotide 41 gtcatagcta tgacacctta 20

1. A compound 8 to 50 nucleobases in length targeted to a nucleic acidmolecule encoding human apolipoprotein(a), wherein said compoundspecifically hybridizes with a nucleic acid molecule encoding humanapolipoprotein(a) and inhibits the expression of humanapolipoprotein(a).
 2. The compound of claim 1 which is an antisenseoligonucleotide.
 3. (Canceled).
 4. The compound of claim 2 wherein theantisense oligonucleotide comprises at least one modifiedinternucleoside linkage.
 5. The compound of claim 4 wherein the modifiedinternucleoside linkage is a phosphorothioate linkage.
 6. The compoundof claim 2 wherein the antisense oligonucleotide comprises at least onemodified sugar moiety.
 7. The compound of claim 6 wherein the modifiedsugar moiety is a 2′-O-methoxyethyl sugar moiety.
 8. The compound ofclaim 2 wherein the antisense oligonucleotide comprises at least onemodified nucleobase.
 9. The compound of claim 8 wherein the modifiednucleobase is a 5-methylcytosine.
 10. The compound of claim 2 whereinthe antisense oligonucleotide is a chimeric oligonucleotide.
 11. Acompound 8 to 50 nucleobases in length which specifically hybridizeswith at least an 8-nucleobase portion of an active site on a nucleicacid molecule encoding human apolipoprotein(a).
 12. A compositioncomprising the compound of claim 1 and a pharmaceutically acceptablecarrier or diluent.
 13. The composition of claim 12 further comprising acolloidal dispersion system.
 14. The composition of claim 12 wherein thecompound is an antisense oligonucleotide.
 15. A method of inhibiting theexpression of human apolipoprotein(a) in cells or tissues comprisingcontacting said cells or tissues with the compound of claim 1 so thatexpression of human apolipoprotein(a) is inhibited.
 16. A method oftreating a human having a disease or condition associated with humanapolipoprotein(a) comprising administering to said human atherapeutically or prophylactically effective amount of the compound ofclaim 1 so that expression of human apolipoprotein(a) is inhibited. 17.The method of claim 16 wherein the condition involves abnormal lipidmetabolism.
 18. The method of claim 16 wherein the condition involvesabnormal cholesterol metabolism.
 19. The method of claim 16 wherein thecondition is atherosclerosis.
 20. The method of claim 16 wherein thedisease is cardiovascular disease.